System and method for processing biological fluid

ABSTRACT

A system for collecting and processing donated blood comprises a first porous medium interposed between a blood collection bag and a satellite bag and a second porous medium interposed between the blood collection bag and another satellite bag. The porous media are leucocyte depletion media. The system may also include one or more of the following: a red cell barrier medium, a separation medium, a gas inlet, and a gas outlet. The system can be used to centrifuge whole blood into one or more components, and includes a means for protecting the system during centrifugation.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 07/609,654, filed Nov. 6, 1990, now U.S. Pat. No.5,100,564, which is incorporated herein by reference.

TECHNICAL FIELD

This invention relates to a system for processing blood donated for thepurpose of therapeutic transfusion of blood components and,particularly, to improved methods and apparatuses for preparing, fromthe donated whole blood, platelet-rich plasma (hereinafter PRP), packedred cells (hereinafter PRC), platelet concentrate (hereinafter PC), andplasma. This invention also relates to a biological fluid processingsystem for processing biological fluid into its various components.

BACKGROUND OF THE INVENTION

The development of plastic blood collection bags has facilitated theseparation of donated whole blood into its various components andanalogous products, thereby making these different blood products (e.g.,platelet concentrates) available as a transfusion product.

With the passage of time and accumulation of research and clinical data,transfusion practices have changed greatly One aspect of currentpractice is that whole blood is rarely administered; rather, patientsneeding red blood cells are given packed red cells, patients needingplatelets are given platelet concentrate, and patients needing plasmaare given plasma.

For this reason, the separation of blood into components has substantialtherapeutic and monetary value. This is nowhere more evident than intreating the increased damage to a patient's immune system caused by thehigher doses and stronger drugs now used during chemotherapy for cancerpatients. These more aggressive chemotherapy protocols are directlyimplicated in the reduction of the platelet content of the blood toabnormally low levels; associated internal and external bleedingadditionally requires more frequent transfusions of PC, and this has putpressure on blood banks to increase the platelet yield per unit ofblood.

A typical component separation procedure used in the United States, thecitrate-phosphate-dextrose-adenine (CPDA-1) system, utilizes a series ofsteps to separate donated blood into three components, each componenthaving substantial therapeutic and monetary value. The proceduretypically utilizes a blood collection bag which is integrally attachedvia flexible tubing to at least one, and preferably two or more,satellite bags. Using centrifugation, whole blood may be separated bydifferential sedimentation into such valuable blood components asplasma, packed red cells (PRC), platelets suspended in clear plasma(platelet-rich plasma, or PRP), platelet concentrate (PC), andcryoprecipitate (which may require extra processing).

A typical whole blood collection and processing procedure may includethe following: (1) A unit of donated whole blood (about 450 ml in UnitedStates, practice) is collected from the donor's vein directly into theblood collection bag which contains the nutrient and anti-coagulantcontaining CPDA-1.

(2) The blood collection bag is centrifuged (slow speed, or "soft-spin"centrifugation) together with its satellite bags, thereby concentratingthe red cells as PRC in the lower portion of the blood collection bagand leaving in the upper portion of the bag a suspension of PRP.

(3) The blood collection bag is transferred, with care not to disturbthe interface between the supernatant PRP layer and the sedimented PRClayer, into a device known as a "plasma extractor." The plasma extractoror expressor typically includes front and back plates; the two platesare hinged together at their lower ends and spring biased toward eachother such that a pressure of about 90 millimeters of mercury isdeveloped within the bag.

With the blood collection bag positioned between the two plates, avalve, seal or a closure in or on the flexible tubing is opened allowingthe supernatant PRP to flow into a first satellite bag. As the PRP flowsout of the blood collection bag, the interface with the PRC rises. Incurrent practice, the operator must closely observe the position of theinterface as it rises and clamp off the connecting tube when, in hisjudgment, as much PRP has been transferred as is possible, withoutallowing red cells to enter the first satellite bag. This is a laborintensive and time consuming operation during which the operator mustvisually monitor the bag and judiciously and arbitrarily ascertain whento shut-off the connecting tube.

The blood collection bag, now containing only PRC, may be detached andstored at 4° C. until required for transfusion into a patient, or avalve or seal in the tubing may be opened so that the PRC may betransferred to a satellite bag using either the pressure generated bythe plasma extractor, or by placing the blood collection apparatus in apressure cuff, or by elevation to obtain gravity flow.

(4) The PRP-containing satellite bag, together with another satellitebag, is then removed from the extractor and centrifuged at an elevated Gforce (high speed or "hard-spin" centrifugation) with the time and speedadjusted so as to concentrate the platelets into the lower portion ofthe PRP bag. When centrifugation is complete, the PRP bag containssedimented platelets in its lower portion and clear plasma in its upperportion.

(5) The PRP bag is then placed in the plasma extractor, and most of theclear plasma is expressed into a satellite bag, leaving the PRP bagcontaining only the sedimented platelets and about 50 ml of plasma; thenin a subsequent step, this platelet composition is dispersed to makeplatelet concentrate (PC). The PRP bag, now containing a PC product, isthen detached and stored for up to five days at 20°-24° C., until neededfor a transfusion of platelets. Multiple units of platelets (e.g., from6-10 donors, if for transfusion into an adult patient) may be pooledinto a single platelet transfusion.

(6) The plasma in the satellite bag may itself be transfused into apatient, or it may be separated by complex processes into a variety ofother valuable products.

Commonly used systems other than CPDA-1 include Adsol, Nutricell, andSAG-M. In these latter systems, the collection bag contains onlyanticoagulant, and the nutrient solution may be preplaced in a satellitebag. This nutrient solution is transferred into the PRC after the PRPhas been separated from the PRC, thereby achieving a higher yield ofplasma and longer storage life for the PRC.

In view of this, there is a growing need for an efficient system andmethod for separating a biological fluid (e.g., whole blood) into itscomponents. Blood bank personnel have responded to the increased needfor blood components by attempting to increase PRC and PC yields in avariety of ways. In separating the PRC and PRP fractions (e.g., step 3above), blood bank personnel have attempted to express more PRP prior tostopping flow from the blood collection bag, but this has often provedto be counterproductive, since the PRP, and the PC subsequentlyextracted from it, are frequently contaminated by red cells, giving apink or red color to the normally light yellow PC. The presence of redcells in PC is so highly undesirable that pink or red PC is frequentlydiscarded, or subjected to recentrifugation, both of which increaseoperating costs and are labor intensive. As a result, blood bankpersonnel must err on the side of caution by stopping the flow of PRPbefore it has been fully expressed. Thus, the PC is uncontaminated, butthe unexpressed plasma, which is valuable, may be wasted.

This reflects another problem when attempting to increase the yield ofindividual blood components. While each component is valuable, anysavings resulting from increasing the yield may be offset by theincreased labor cost, if the operator of the processing system mustcontinuously and carefully monitor the system to increase the yield.

The devices and methods of this invention alleviate the above-describedproblems and, in addition, provide a higher yield of superior qualityPRC and PC.

The separation of the various blood components using centrifugation isattended by a number of other problems. For example, when PRP iscentrifuged to obtain a layer consisting principally of plateletsconcentrated at the bottom of the PRP-containing bag, e.g., step 4above, the platelets so concentrated tend to form a dense aggregatewhich must be dispersed in plasma to form platelet concentrate. Thedispersion step is usually carried out by gentle mixing, for example, byplacing the bag on a moving table which rotates with a precessing tiltedmotion. This mixing requires several hours, a potentially undesirabledelay, and is believed by many researchers to produce a partiallyaggregated platelet concentrate. It is further believed that theplatelets may be damaged by the forces applied during centrifugation.

Finally, a problem attendant with the separation of various bloodcomponents using a multiple bag system and centrifugation is that highlyvaluable blood components become trapped in the conduits connecting thevarious bags and in the various devices that may be used in the system.

Conventional processing and storage techniques may also presentproblems. For example, air, in particular oxygen, present in storedblood and blood components, or in the storage container, may lead to animpairment of the quality of the blood components, and may decreasetheir storage life. More particularly, oxygen may be associated with anincreased metabolic rate (during glycolysis), which may lead todecreased storage life, and decreased viability and function of wholeblood cells. For example, during storage red blood cells metabolizeglucose, producing lactic and pyruvic acids. These acids decrease the pHof the medium, which in turn decreases metabolic functions. Furthermore,the presence of air or gas in the satellite bag may present a risk whena patient is transfused with a blood component. For example, as littleas 5 ml of air or gas may cause severe injury or death. Despite thedeleterious effect of oxygen on storage life and the quality of bloodand blood components, the prior art has not addressed the need to removegases from blood processing systems during collection and processing.

In addition to the above-listed components, whole blood contains whiteblood cells (known collectively as leucocytes) of various types, ofwhich the most important are granulocytes and lymphocytes. White bloodcells provide protection against bacterial and viral infection. Thetransfusion of blood components which have not been leucocyte-depletedis not without risk to the patient receiving the transfusion. Some ofthese risks are detailed in U.S. Pat. No. 4,923,620, and in U.S. Pat.No. 4,880,548, which are incorporated herein by reference.

In the above described centrifugal method for separating blood into thethree basic fractions, the leucocytes are present in substantialquantities in both the packed red cells and platelet-rich plasmafractions. It is now generally accepted that it is highly desirable toreduce the leucocyte concentration of these blood components to as low alevel as possible. While there is no firm criterion, it is generallyaccepted that many of the undesirable effects of transfusion would bereduced if the leucocyte content were reduced by a factor of about 100or more prior to administration to the patient. This approximatesreducing the average total content of leucocytes in a single unit of PRCto less than about 1×10⁶, and in a unit of PRP or PC to less than about1×10⁵. Devices which have previously been developed in attempts to meetthis objective have been based on the use of packed fibers, and havegenerally been referred to as filters. However, it would appear thatprocesses utilizing filtration based on separation by size cannotsucceed for two reasons. First, leucocytes can be larger than about 15μm (e.g., granulocytes and macrocytes) to as small as 5 to 7 μm (e.g.,lymphocytes). Together, granulocytes and lymphocytes represent the majorproportion of all of the leucocytes in normal blood. Red blood cells areabout 7 μm in diameter, i.e., they are about the same size aslymphocytes, one of the two major classes of leucocytes which must beremoved. Secondly, all of these cells deform so that they are able topass through much smaller openings than their normal size. Accordingly,it has been widely accepted that removal of leucocytes is accomplishedmainly by adsorption on the internal surfaces of porous media, ratherthan by filtration.

Leucocyte depletion is particularly important with respect to a bloodcomponent such as PC. Platelet concentrates prepared by the differentialcentrifugation of blood components will have varying levels of leucocytecontamination related to the time and to the magnitude of the forcedeveloped during centrifugation. The level of leucocyte contamination inunfiltered conventional platelet preparations of 6 to 10 pooled units isgenerally at a level of about 5×10⁸ or greater. It has been demonstratedthat leucocyte removal efficiencies of 81 to 85% are sufficient toreduce the incidence of febrile reactions to platelet transfusions.Several other recent studies report a reduction in alloimmunization andplatelet refractoriness at levels of leucocyte contamination below about1×10⁷ per unit. For a single unit of PC averaging a leucocytecontamination level (under current practice) of about 7×10⁶ ;leucocytes, the goal after filtration is less than 1×10⁶ leucocytes. Theexisting studies, therefore, suggest the desirability of at least a twolog (99%) reduction of leucocyte contamination. More recent studiessuggest that a three log (99.9%) or even a four log (99.99%) reductionwould be significantly more beneficial.

An additional desirable criterion is to restrict platelet loss to about15% or less of the original platelet concentration. Platelets arenotorious for being "sticky", an expression reflecting the tendency ofplatelets suspended in blood plasma to adhere to any non-physiologicalsurface to which they are exposed. Under many circumstances, they alsoadhere strongly to each other.

In any system which depends upon filtration to remove leucocytes from aplatelet suspension, there will be substantial contact between plateletsand the internal surfaces of the filter assembly. The filter assemblymust be such that the platelets have minimal adhesion to, and are notsignificantly adversely affected by contact with, the filter assembly'sinternal surfaces.

If the leucocyte depletion device comprises a porous structure,microaggregates, gels, fibrin, fibrinogen and fat globules tend tocollect on or within the pores, causing blockage which inhibits flow.Conventional processes, in which the filter for depleting leucocytesfrom PRC is pre-conditioned by passing saline through the filterassembly with or without a post-filtration saline flush, are undesirablebecause the liquid content of the transfusion is unduly increased, thuspotentially overloading the patient's circulatory system with liquid. Anobjective of an embodiment of this invention is a leucocyte depletiondevice which removes leucocytes and these other elements with highefficiency and without clogging, requires no preconditioning prior toprocessing PRC derived from freshly drawn blood, and does not requirepost-filtration flushing to reclaim red cells remaining in the filter.

Because of the high cost and limited availability of blood components, adevice comprising a porous medium used to deplete leucocytes frombiological fluid should deliver the highest possible proportion of thecomponent present in the donated blood. An ideal device for theleucocyte depletion of PRC or PRP would be inexpensive, relativelysmall, and be capable of rapidly processing blood components obtainedfrom about one unit or more of biological fluid (e.g., donated wholeblood), in, for example, less than about one hour. Ideally, this devicewould reduce the leucocyte content to the lowest possible level, whilemaximizing the yield of a valuable blood component while minimizing anexpensive, sophisticated, labor intensive effort by the operator of thesystem. The yield of the blood component should be maximized while atthe same time delivering a viable and physiologically activecomponent--e.g., by minimizing damage due to centrifugation, and/or thepresence of air or gas. It may also be preferable that the PRC porousmedium be capable of removing platelets, as well as fibrinogen, fibrinstrands, tiny fat globules, and other components such as microaggregateswhich may be present in whole blood.

DEFINITIONS

The following definitions are used in reference to the invention:

(A) Blood Product or Biological Fluid: anti-coagulated whole blood(AWB); packed red cells obtained from AWB; platelet-rich plasma (PRP)obtained from AWB; platelet concentrate (PC) obtained from AWB or PRP;plasma obtained from AWB or PRP; red cells separated from plasma andresuspended in physiological fluid; and platelets separated from plasmaand resuspended in physiological fluid. Blood product or biologicalfluid also includes any treated or untreated fluid associated withliving organisms, particularly blood, including whole blood, warm orcold blood, and stored or fresh blood; treated blood, such as blooddiluted with a physiological solution, including but not limited tosaline, nutrient, and/or anticoagulant solutions; one or more bloodcomponents, such as platelet concentrate (PC), platelet-rich plasma(PRP), platelet-free plasma, platelet-poor plasma, plasma, or packed redcells (PRC); analogous blood products derived from blood or a bloodcomponent or derived from bone marrow. The biological fluid may includeleucocytes, or may be treated to remove leucocytes. As used herein,blood product or biological fluid refers to the components describedabove, and to similar blood products or biological fluids obtained byother means and with similar properties. In accordance with theinvention, each of these blood products or biological fluids isprocessed in the manner described herein.

(B) Unit of Whole Blood: Blood banks in the United States commonly drawabout 450 milliliters (ml) of blood from the donor into a bag whichcontains an anticoagulant to prevent the blood from clotting. However,the amount drawn differs from patient to patient and donation todonation. Herein the quantity drawn during such a donation is defined asa unit of whole blood.

(C) Unit of Packed Red Cells (PRC), Platelet-rich Plasma (PRP) orPlatelet Concentrate (PC): As used herein, a "unit" is defined by theUnited States, practice, and a unit of PRC, PRP, PC, or of red cells orplatelets in physiological fluid or plasma, is the quantity derived fromone unit of whole blood. It may also refer to the quantity drawn duringa single donation. Typically, the volume of a unit varies. For example,the volume of a unit of PRC varies considerably depending on thehematocrit (percent by volume of red cells) of the drawn whole blood,which is usually in the range of about 37% to about 54%. The concomitanthematocrit of PRC, which varies over the range from about 50% to over80%, depends in part on whether the yield of one or another bloodproduct is to be minimized. Most PRC units are in the range of about 170to about 350 ml, but variation below and above these figures is notuncommon. Multiple units of some blood components, particularlyplatelets, may be pooled or combined, typically by combining 6 or moreunits.

(D) Plasma-Depleted Fluid: A plasma-depleted fluid refers to anybiological fluid which has had some quantity of plasma removedtherefrom, e.g., the platelet-rich fluid obtained when plasma isseparated from PRP, or the fluid which remains after plasma is removedfrom whole blood.

(E) Porous medium: refers to the porous medium through which one or moreblood components or biological fluids pass. The PRC porous mediumdepletes leucocytes from the packed red cell component. The platelet orPRP porous medium refers generically to any one of the media whichdeplete leucocytes from the non-PRC blood components, i.e., from PRP orfrom PC. The red cell barrier medium blocks the passage of red cells anddepletes leucocytes from PRP to a greater or lesser degree whileallowing the passage of platelets.

As noted in more detail below, the porous medium for use with PRC may beformed from any natural or synthetic fiber (or from other materials ofsimilar surface area and pore size) compatible with blood. The porousmedium may remain untreated. Preferably, the critical wetting surfacetension (CWST) of the porous medium is within a certain range, as notedbelow and as dictated by its intended use. The pore surfaces of themedium may be modified or treated in order to achieve the desired CWST.For example, the CWST of a PRC porous medium is typically above about 53dynes/cm.

The porous medium for use with PRP may be formed from any natural orsynthetic fiber or other porous material compatible with blood. Theporous medium may remain untreated. Preferably, the CWST and zetapotential of the porous medium are within certain ranges, as disclosedbelow and as dictated by its intended use. For example, the CWST of aPRP porous medium is typically above about 70 dynes/cm.

The porous media according to the invention may be connected to aconduit interposed between the containers, and may be positioned in ahousing which in turn can be connected to the conduit. As used herein,filter assembly refers to the porous medium positioned in a suitablehousing. An exemplary filter assembly may include a leucocyte depletionassembly or device or a red cell barrier assembly or device. Abiological fluid processing system, such as a blood collection andprocessing system, may comprise porous media, preferably as filterassemblies. Preferably, the porous medium forms an interference fit atits edges when assembled into the housing.

The porous medium may be configured as a flat sheet, a corrugated sheet,a web, or a membrane. The porous medium may be pre-formed, andconfigured as hollow fibers, although it is not intended that theinvention should be limited thereby.

(F) Separation Medium: A separation medium refers to a porous mediumeffective for separating one component of a biological fluid fromanother component. The separation media according to the invention aresuitable for passing at least one component of the blood product orbiological fluid, particularly plasma, therethrough, but not othercomponents of the blood product or biological fluid, particularlyplatelets and/or red cells.

As noted in more detail below, the separation medium for use with abiological fluid may be formed from any natural or synthetic fiber orfrom a porous or permeable membrane (or from other materials of similarsurface area and pore size) compatible with a biological fluid. Thesurface of the fibers or membrane may be unmodified or may be modifiedto achieve a desired property. Although the separation medium may remainuntreated, the fibers or membrane are preferably treated to make themeven more effective for separating one component of a biological fluid,e.g., plasma, from other components of a biological fluid, e.g.,platelets or red cells. The separation medium is preferably treated inorder to reduce or eliminate platelet adherence to the medium. Anytreatment which reduces or eliminates platelet adhesion is includedwithin the scope of the present invention. Furthermore, the medium maybe surface modified as disclosed in U.S. Pat. No. 4,880,548,incorporated herein by reference, in order to increase the criticalwetting surface tension (CWST) of the medium and to be less adherent ofplatelets. Defined in terms of CWST, a preferred range of CWST for aseparation medium according to the invention is above about 70 dynes/cm,more preferably above about 90 dynes/cm. Also, the medium may besubjected to gas plasma treatment in order to reduce platelet adhesion.Preferably, the critical wetting surface tension (CWST) of theseparation medium is within a certain range, as noted below and asdictated by its intended use. The pore surfaces of the medium may bemodified or treated in order to achieve the desired CWST.

The separation medium may be pre-formed, multi-layered, and/or may betreated to modify the surface of the medium. If a fibrous medium isused, the fibers may be treated either before or after forming thefibrous lay-up. It is preferred to modify the fiber surfaces beforeforming the fibrous lay-up because a more cohesive, stronger product isobtained after hot compression to form an integral filter element. Theseparation medium is preferably pre-formed.

The separation medium may be configured in any suitable fashion, such asa flat sheet, a corrugated sheet, a web, hollow fibers, or a membrane.

(G) Voids volume is the total volume of all of the pores within a porousmedium. Voids volume is expressed hereinafter as a percentage of theapparent volume of the porous medium.

(H) Measurement of fiber surface area and of average fiber diameter: Inaccordance with the invention, a useful technique for the measurement offiber surface area, for example by gas adsorption, is generally referredto as the "BET" measurement. The surface area of melt blown webs can beused to calculate average fiber diameter, using PBT as an example:##EQU1## where L=total length in cm of 1 gram of fiber,

d=average fiber diameter in centimeters, and

A_(f) =fiber surface area in cm² /g.

If the units of d are micrometers, the units of A_(f) become M² /g(square meters/gram), which will be used hereinafter.

(I) Critical Wetting Surface Tension: As disclosed in U.S. Pat. No.4,880,548, the CWST of a porous medium may be determined by individuallyapplying to its surface a series of liquids with surface tensionsvarying by 2 to 4 dynes/cm and observing the absorption ornon-absorption of each liquid over time. The CWST of a porous medium, inunits of dynes/cm, is defined as the mean value of the surface tensionof the liquid which is absorbed and that of the liquid of neighboringsurface tension which is not absorbed within a predetermined amount oftime. The absorbed and non-absorbed values depend principally on thesurface characteristics of the material from which the porous medium ismade and secondarily on the pore size characteristics of the porousmedium.

Liquids with surface tensions lower than the CWST of a porous mediumwill spontaneously wet the medium on contact, and, if the pores of themedium are interconnected, liquid will flow through the medium readily.Liquids with surface tensions higher than the CWST of the porous mediummay not flow at all at low differential pressures, or may flow unevenlyat sufficiently high differential pressures to force the liquid throughthe porous medium. In order to achieve adequate priming of a fibrousmedium with a liquid such as blood, the fibrous medium preferably has aCWST in the range of about 53 dynes/cm or higher.

For the porous medium which is used to process PRC, it is preferred thatthe CWST be held within a range somewhat above the CWST of untreatedpolyester fiber (52 dynes/cm), for example, above about 53 dynes/cm,more preferably, above about 60 dynes/cm. For the porous medium which isused to process PRP, it is preferred that the CWST be held within arange above about 70 dynes/cm.

(J) General procedure for measuring zeta potential: Zeta potential wasmeasured using a sample cut from a 1/2 inch thick stack of webs.

The zeta potential was measured by placing the sample in an acrylicfilter holder which held the sample snugly between two platinum wirescreens 100×100 mesh (i.e., 100 wires in each direction per inch). Themeshes were connected, using copper wire, to the terminals of a TriplettCorporation model 3360 Volt-Ohm Meter, the mesh on the upstream side ofthe sample being connected to the positive terminal of the meter. ApH-buffered solution was flowed through the sample using a differentialpressure of 45 inches of water column across the filter holder and theeffluent was collected. For measurements at pH 7, a buffered solutionwas made by adding 6 ml pH 7 buffer (Fisher Scientific Co. catalognumber SB108-500) and 5 ml pH 7.4 buffer (Fisher Scientific Co. catalognumber SB110-500) to 1 liter pyrogen-free deionized water. Formeasurements at pH 9, a buffered solution was made by adding 6 ml pH 9buffer (Fisher Scientific Co. catalog number SB114- 500) and 2 ml pH 10buffer (Fisher Scientific Co. catalog number SB116-500) to 1 literpyrogen-free deionized water. The electrical potential across the filterholder was measured during flow (it required about 30 seconds of flowfor the potential to stabilize) and was corrected for cell polarizationby subtracting from it the electrical potential measured when flow wasstopped. During the period of flow the pH of the liquid was measuredusing a Cole-Parmer model J-5994-10 pH meter fitted with an in-linemodel J-5993-90 pH probe. The conductivity of the liquid was measuredusing a Cole-Parmer model J-1481-60 conductivity meter fitted with amodel J-1481-66 conductivity flow cell. Then the polarity of the voltmeter was reversed, and the effluent was flowed backwards through thefilter holder using a differential pressure of 45 inches of watercolumn. As in the first instance the electrical potential measuredduring flow was corrected for cell polarization by subtracting from itthe electrical potential measured after flow was stopped. The average ofthe two corrected potentials was taken as the streaming potential.

The zeta potential of the medium was derived from the streamingpotential using the following relationship (J. T. Davis et al.,Interfacial Phenomena. Academic Press, New York, 1963): ##EQU2## where^(n) is the viscosity of the flowing solution, D is its dielectricconstant, λ is its conductivity, E_(S) is the streaming potential and Pis the pressure drop across the sample during the period of flow. Inthese tests the quantity 4 π^(n) /DP was equal to 0.800.

(K) Tangential flow filtration: As used herein, tangential flowfiltration refers to passing or circulating a biological fluid in agenerally parallel or tangential manner to the surface of the separationmedium.

SUMMARY OF THE INVENTION

In the devices and methods of this invention, leucocyte depletion of abiological fluid (e.g., PRC or PRP) is carried out at the time ofprocessing, which, in the United States, is generally within about 6 to8 hours of the time the blood is drawn. Thus, as a biological fluid istransferred from the bag in which it is contained, leucocytes areremoved by the appropriate porous medium, and leucocyte-depletedbiological fluid is collected in the satellite bag. In accordance withthe invention, a system is provided whereby a biological fluid such aswhole blood is processed to form PRP and PRC. PRP is leucocyte depletedby interposing between the blood collection bag and a first satellitebag at least one porous medium for depleting leucocytes from PRP; PRC isleucocyte depleted by interposing between the blood collection bag and asecond satellite bag at least one porous medium for removing leucocytesfrom PRC.

The invention also comprises a centrifugation system wherein one (orboth) of the interposed leucocyte depletion filter assemblies is (are)cooperatively arranged with a centrifuge bucket in a manner such thatthe filter assembly, the porous medium in the filter assembly, and theblood bags are not damaged by the very large forces generated during thecentrifugation process.

Processes and systems according to the invention may also include a redcell barrier medium that allows the passage of one component of thebiological fluid, but prevents the passage of another component throughthe medium, thereby eliminating the need for continuous monitoring by anoperator and increasing the efficiency with which a biological fluidsuch as whole blood is separated into one or more components.

Additionally, processes and systems according to the invention mayinclude a gas outlet that allows gas that may be present in the systemout of the system.

Processes and systems according to the invention may also include a gasinlet that allows gas into the system to recover a biological fluid thatmay be entrapped or retained during processing.

The invention also involves the treatment of a biological fluid tonon-centrifugally separate at least one component from the biologicalfluid, e.g., treating PRP to obtain plasma and PC, or separating plasmafrom whole blood. Processes and devices according to the inventionutilize a separation medium that allows the passage of one component ofthe biological fluid, such as plasma, but prevents passage of othercomponents, such as platelets or red cells, through the medium, therebyeliminating the need for "hard-spin" centrifugation as a processingstep. Tangential flow of a biological fluid parallel to the upstreamsurface of the separating medium permits the passage of plasma throughthe medium, while reducing the tendency for cellular components orplatelets to adhere to the surface of the medium, thus assisting in theprevention of passage of platelets through the separation medium. Thehydrodynamics of flow parallel to a surface are indeed believed to besuch that during flow parallel to the surface, platelets develop a spinwhich causes them to be recovered from the surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an embodiment of a biological fluid processing systemaccording to the invention, whereby biological fluid is separated intocomponents by centrifugal separation.

FIG. 2 is another embodiment of a biological fluid processing systemaccording to the invention, including a non-centrifugal separationdevice.

FIG. 3 is an embodiment of the invention which incorporates a gas inletand a gas outlet.

FIG. 4 is an exploded perspective view of one embodiment of a filterassembly, a centrifuge bucket, and a holder to properly position thefilter assembly on the bucket.

FIG. 5 is an elevation of an embodiment of the present invention.

FIG. 6 is a cross-section of an embodiment of the invention, showing thefirst fluid flow path in a separation device according to the invention.

FIG. 7 is a section of FIG. 6, along A--A.

FIG. 8 is a section of FIG. 6, along B--B.

FIG. 9 is a cross-section of an embodiment of the invention, showing thesecond fluid flow path in a separation device according to theinvention.

FIG. 10 is a section of FIG. 9, along C--C.

FIG. 11 is a section of FIG. 9, along D--D.

SPECIFIC DESCRIPTION OF THE INVENTION

The present invention involves a biological fluid, preferably blood,collection and processing assembly comprising a first container and asecond container, and a conduit interconnecting the first container withthe second container; and at least one third container and a conduitinterconnecting the first container with the third container; and havinginterposed between the first container and a second container, at leastone first porous medium; and having interposed between the firstcontainer and a third container, at least one second porous medium. Thefirst porous medium may be a leucocyte depletion medium, a red cellbarrier medium, an assembly comprising a leucocyte depletion medium anda red cell barrier medium, or combinations thereof. The second porousmedium may be a leucocyte depletion medium which may, optionally,include a microaggregate filter element and/or a gel prefilter element.As shown in more detail below, the assembly may also include additionalcontainers, porous media, and conduits interconnecting the containersand porous media.

In another embodiment of the invention, the blood collection andprocessing assembly comprises containers interconnected with a conduit,and a porous medium interposed in the conduit for depleting leucocytesfrom PRC wherein the porous medium has a CWST greater than about 53dynes/cm.

In another embodiment of the invention, the blood collection andprocessing assembly comprises containers interconnected with a conduit,and a porous medium interposed in the conduit for depleting leucocytesfrom PRP wherein the porous medium has a CWST greater than about 70dynes/cm.

The invention also involves a biological fluid processing systemcomprising a first container; a first porous medium comprising a redcell barrier medium communicating with the first container, and defininga first flow path; and a second porous medium comprising a leucocytedepletion medium communicating with the first container, and defining asecond flow path. As shown in more detail below, the system may alsoinclude additional containers, flow paths, and porous media.

The invention also involves a method for collecting and processing bloodcomprising collecting whole blood in a container; centrifuging the wholeblood; passing the supernatant layer of the centrifuged blood through afirst porous medium, the first porous medium comprising at least one ofa leucocyte depletion medium, a red cell barrier medium, and a combinedleucocyte depletion red cell barrier medium; and passing the sedimentlayer of the centrifuged blood through a second porous medium, thesecond porous medium comprising a leucocyte depletion medium.

The invention also involves a method for processing a biological fluidcomprising expressing a biological fluid from a first container to afirst porous medium comprising a red cell barrier medium; and expressinga biological fluid from the first container to a second porous medium.As shown in more detail below, the method may also include processingthe fluid through additional containers, flow paths, and porous media.

An exemplary biological fluid collection and processing system is shownin FIG. 1. The biological fluid processing system is generally denotedas 10. It may comprise a first container or collection bag 11; a needleor cannula 1 adapted to be inserted into the donor; an optional red cellbarrier assembly 12; a first leucocyte depletion assembly 13; a secondcontainer (first satellite bag) 41; an optional fourth container (thirdsatellite bag) 42; a second leucocyte depletion assembly 17; and a thirdcontainer (second satellite bag) 18. Each of the assemblies orcontainers may be in fluid communication through tubing, preferablyflexible tubing, 20, 21, 25, 26, 27 or 28. The first leucocyte depletionassembly preferably includes a porous medium for passing PRP; the secondleucocyte depletion assembly preferably includes a porous mediumsuitable for passing PRC. A seal, valve, clamp, or transfer leg closureor cannula (not illustrated) may also be positioned in or on the tubingor in the collection and/or satellite bags. The seal (or seals) isopened when fluid is to be transferred between bags.

In another exemplary configuration, the blood processing system shown inFIG. 2 is the same as the exemplary system shown in FIG. 1, except thatthe portion of the system downstream of leucocyte depletion assembly 13includes a separation assembly 14, preferably a non-centrifugalseparation assembly. Separation assembly 14 is in fluid communicationwith the first leucocyte depletion assembly 13 through tubing 22 andcommunicates with satellite bags 15, 16 through tubing 23, 24.

In another exemplary configuration, shown in FIG. 3, the invention mayalso comprise at least one gas inlet 51, 53 and/or at least one gasoutlet 52, 54. The system of FIG. 3 includes a first container orcollection bag 11 in fluid communication with an optional red cellbarrier assembly 12, gas inlet 53, a leucocyte depletion assembly 13,and gas outlet 54. First container 11 may also be in fluid communicationwith a gas inlet 51, a leucocyte depletion assembly 17, and a gas outlet52. As shown in more detail below, the assembly may also includeadditional containers, flow paths, and porous media.

Any number and combinations of assemblies, porous media, containers, andconduits are suitable. One skilled in the art will recognize that theinvention as described here may be reconfigured into differentcombinations, which are included within the scope of the invention.

Each of the components of the assembly will now be described in moredetail below.

The containers which are used in the biological fluid processingassembly may be constructed of any material compatible with a biologicalfluid, such as whole blood or a blood component, and capable ofwithstanding a centrifugation and sterilization environment. A widevariety of these containers are already known in the art. For example,blood collection and satellite bags are typically made from plasticizedpolyvinyl chloride, e.g. PVC plasticized with dioctylphthalate,diethylhexylphthalate, or trioctyltrimellitate. The bags may also beformed from polyolefin, polyurethane, polyester, and polycarbonate.

As used herein, the tubing may be any conduit or means which providesfluid communication between the containers, and is typically made fromthe same flexible material as is used for the containers, preferablyplasticized PVC. The tubing may extend into the interior of thecontainer, and may be used as a siphon, for example. There may be anumber of tubes providing fluid communication to any individualcontainer, and the tubes may be oriented in a number of ways. Forexample, there may be at least two tubes oriented at the top of thecollection bag, or at the bottom of the bag, or a tube at each end ofthe bag.

Additionally, the tubes, assemblies, porous media, and containers, maybe oriented to define different flow paths. For example, when wholeblood is processed, the PRP may flow along a first flow path, e.g.,through the red cell barrier assembly (if present), a PRP leucocytedepletion assembly, and into a satellite bag (e.g., a second container).Similarly, the PRC may flow along a second flow path, e.g., through thePRC leucocyte depletion assembly, and into a satellite bag (e.g., athird container). Since independent flow paths may be present,biological fluids (e.g., PRP and PRC) may flow concurrently, orsequentially.

A seal, valve, clamp, transfer leg closure, or the like is typicallylocated in or on the tubing. It is intended that the present inventionis not limited by the type of material used to construct the containersor the conduit which connects the containers.

The composition of the various porous media will depend in part on thefunction desired, e.g., red blood cell blockage or leucocyte depletion.A preferred composition of the various porous media is a mat or webcomposed of fibers, which are preferably thermoplastic. The fibers ofthe porous media may comprise any fiber compatible with biologicalfluid, and may be either natural or synthetic fibers. In accordance withthe invention, the fibers are preferably treated or modified in order toachieve or increase the CWST. For example, the fibers may be surfacemodified to increase the critical wetting surface tension (CWST) of thefibers. For example, the treated or untreated fibers used in the PRCporous medium preferably have a CWST above about 53 dynes/cm; for thePRP porous medium, above about 70 dynes/cm. Also, the fibers may bebonded, fused, or otherwise fixed to one another, or they may bemechanically entwined. Other porous media, for example, open cell foamedplastics, surface modified as noted above, may be similarly used.

While the porous media can be produced from any material compatible withbiological fluid, practical considerations dictate that consideration begiven first to the use of commercially available materials. The porousmedia of this invention may be preferably formed, for example, from anysynthetic polymer capable of forming fibers and of serving as asubstrate for grafting. Preferably, the polymer should be capable ofreacting with at least one ethylenically unsaturated monomer under theinfluence of ionizing radiation without the matrix being significantlyor excessively adversely affected by the radiation. Suitable polymersfor use as the substrate include, but are not limited to, polyolefins,polyesters, polyamides, polysulfones, acrylics, polyacrylonitriles,polyaramides, polyarylene oxides and sulfides, and polymers andcopolymers made from halogenated olefins and unsaturated nitriles.Examples include, but are not limited to, polyvinylidene fluoride,polyethylene, polypropylene, cellulose acetate, and Nylon 6 and 66.Preferred polymers are polyolefins, polyesters, and polyamides. The mostpreferred polymer is polybutylene terephthalate (PBT).

Surface characteristics of a fiber may remain unmodified, or can bemodified by a number of methods, for example, by chemical reactionincluding wet or dry oxidation; by coating the surface by depositing apolymer thereon; or by grafting reactions wherein the substrate or fibersurface is activated prior to or during wetting of the fiber surface bya monomer solution by exposure to an energy source such as heat, a Vander Graff generator, ultraviolet light, or to various other forms ofradiation; or by subjecting the fibers to gas plasma treatment. Apreferred method is a grafting reaction using gamma-radiation, forexample, from a cobalt source.

An exemplary radiation grafting technique employs at least one of avariety of monomers each comprising an ethylene or acrylic moiety and asecond group, which is preferred to be a hydrophilic group (e.g.,--COOH, or --OH). Grafting of the fibrous medium may also beaccomplished by compounds containing an ethylenically unsaturated group,such as an acrylic moiety, combined with a hydroxyl group, preferablymonomers such as hydroxyethyl methacrylate (HEMA), or acrylic acid. Thecompounds containing an ethylenically unsaturated group may be combinedwith a second monomer such as methyl acrylate (MA), methyl methacrylate(MMA), or methacrylic acid (MAA). MA or MMA are preferably incorporatedinto the porous medium used to treat PRC, and MAA is preferablyincorporated into the porous medium used to treat the PRP. Preferably,the MAA to HEMA monomer weight ratio in the modifying mixture may bebetween about 0.01:1 and about 0.5:1; preferably, the MA or MMA to HEMAmonomer weight ratio in the modifying mixture may be between about0.01:1 and about 0.4:1. Use of HEMA contributes to a very high CWST.Analogues with similar functional characteristics may also be used tomodify the surface characteristics of fibers.

It has been observed that porous media, surface treated using somegrafting monomers or combinations of monomers, behave differently withrespect to the span between the surface tension of the liquid which isabsorbed and the surface tension of the liquid which is not absorbedwhen determining the CWST. This span can vary from less than 3 to asmuch as 20 or more dynes/cm. Preferably, the media has a span betweenthe absorbed and non-absorbed values of about 5 or fewer dynes/cm. Thischoice reflects the greater precision with which the CWST can becontrolled when narrower spans are selected, albeit media with widerspans may also be used. The use of the narrower span is preferred inorder to improve product quality control.

Radiation grafting may increase fiber-to-fiber bonding in a fibrousmedium. Consequently, a fibrous medium which exhibits little or nofiber-to-fiber bonding in an untreated state may exhibit significantfiber-to-fiber bonding after the fibers have been radiation grafted toincrease the CWST of the medium.

For the porous media for use with a biological fluid such as PRP, apreferred range for the CWST of the fiber is preferably above about 70dynes/cm, typically about 70 to 115 dynes/cm; a more preferred range is90 to 100 dynes/cm, and a still more preferred range is 93 to 97dynes/cm. A preferred range for the zeta potential (at the pH of plasma(7.3)) is about -3 to about -30 millivolts, a more preferred range isabout -7 to about -20 millivolts, and a still more preferred range isabout -10 to about -14 millivolts.

In packed red cells, as well as in whole blood, the red cells aresuspended in blood plasma, which has a surface tension of about 73dynes/cm. For the depletion of the leucocyte content of PRC, a CWSTgreater than about 53 dynes/cm is desirable. The CWST may typically beabove from about 53 dynes/cm to about 115 dynes/cm, but the inventionshould not be limited thereby. A more preferable CWST is above about 60dynes/cm, and a still more preferable CWST is from about 62 dynes/cm toless than about 90 dynes/cm.

If desired, the flow rate of biological fluid through the filter can beregulated to obtain a total flow period of about 10 to 40 minutes byselecting the appropriate element diameter, element thickness, fiberdiameter, and density, and/or by varying the diameter of the tube eitherupstream or downstream of the filter, or both up and downstream. Atthese flow rates, leucocyte depletion efficiency in excess of 99.9% maybe achieved. If PRP is the biological fluid being processed, theselevels of efficiency may result in a PC product with less than about0.1×10⁶ leucocytes per unit of PC compared with the target of less thanabout 1×10⁶.

The leucocyte depleting PRC porous medium is primarily intended for usewith PRC obtained from donated blood within about 8 hours of the timethe blood was drawn. It may also be used to filter PRC which has beenstored at 4° C. for up to several weeks, but, since the risk of cloggingduring filtration increases with storage age, the risk can be reduced,by for example, using pre-filters preceding the media described herein.

A PRC porous medium of the invention can be made to have a wide range ofefficiencies for leucocyte depletion. If the porous medium is composedof 2.6 μm fibers and weighs about ##EQU3## where ρ=fiber density,grams/cc and V=voids volume, %,

then when used for leucocyte depletion of PRC, the log of theefficiency, defined as the ratio of the influent leucocyte concentrationto the effluent leucocyte concentration, may be calculated from theformula ##EQU4## In most applications, it is desirable to keep the timeof flow of a unit of PRC through the porous medium when pressurized toabout 30 to 300 mm of Hg to less than about 30 to 40 minutes; in orderto achieve this flow rate, the device should preferably be configured toa flow area of about 30 to 60 cm².

For example, an 8.63 cm diameter (area=58.5 cm²) porous medium madeusing 7.7 grams of 2.6 μm diameter 1.38 g/cm³ density fiber with a voidsvolume of 76.5%, would meet the requirements of equation (3), and itsleucocyte depletion efficiency, in accordance with equation (4) would belog 6. Thus, if the influent concentration were 10⁹ leucocytes/unit,then the effluent concentration would be ##EQU5## Similarly, if madewith V=88.2% using 2.6 μm diameter fibers of density 1.38 g/cc, theweight of the porous medium would be, per equation (3): ##EQU6## and thelog of the efficiency would be, per equation (4): ##EQU7## Thus, if theinfluent leucocyte concentration were 10⁹ per unit of PRC, the effluentconcentration would be ##EQU8## Equations (3) and (4) are applicable toa voids volume range of about 73 to 88.5%, which spans the efficiencyrange from about log 3 to log 7.

Equations (3) and (4) provide very useful guidelines for designing andbuilding optimal or near to optimal PRC filters with limitedexperimentation; however, a person familiar with the art will recognizethat variations from these formulae and modifications to the porousmedia can be made to produce useful products. Exemplary modificationsand their effect on the performance characteristics of the porous mediaare set out below:

    ______________________________________                                        Desired Filter     Changes from                                               Characteristics    Equations (3) and (4)                                      ______________________________________                                        Increased leucocyte                                                                              Reduce fiber diameter.sup.(1)                              depletion efficiency                                                                             Increase weight of                                                            fiber                                                                         Reduce voids volume                                        Decrease probability of                                                                          Increase filter element                                    clogging           area                                                                          Provide prefiltration                                                         Increase voids volume                                      Decrease internal hold-                                                                          Decrease voids volume.sup.(2)                              up volume          Eliminate                                                                     prefiltration.sup.(2)                                                         Use finer fiber.sup.(1)                                    Increase flow rate of                                                                            Process the blood such                                     the PRC            that the PRC has lower                                                        hematocrit, hence lower                                                       viscosity                                                                     Use higher head when                                                          filtering                                                                     Increase filter area                                                          with concomitant                                                              reduction of thickness                                                        Increase filter element                                                       voids volume                                               Withstand higher   Reduce element voids                                       applied differential                                                                             volume                                                     pressure           Use coarser fiber (at                                                         the cost of reduced                                                           efficiency)                                                                   Use fiber with higher                                                         modulus                                                    ______________________________________                                         .sup.(1) Use of too small a fiber diameter may result in collapse of the      filter element at normal working differential pressure.                       .sup.(2) May result in excessively long filtration times, or complete         clogging prior to completion of a transfusion.                           

RED CELL BARRIER MEDIUM

Red cell barrier assemblies made in accordance with an embodiment of theinvention and which are, for example, interposed between the bloodcollection bag and the PRP bag, will generally remove about 85% to 99%or more of the incident leucocytes, a removal rate that may not besufficient to consistently achieve a residual leucocyte count of lessthan 10⁶ leucocytes per unit of PC. A principal function of thisassembly, however, is to act as an automatic "valve" during thedecantation process by instantly stopping the flow of a biological fluidsuch as the supernatant layer (e.g., PRP), at the moment that red cellscontact the porous medium comprising porous media. The mechanism of thisvalve-like action is not well understood, but it may reflect aggregationof the red cells as they reach the porous surface, forming a barrierwhich prevents or blocks further flow of the supernatant layer throughthe porous medium.

Aggregation of red cells on contact with the porous filter appears to berelated to the CWST and/or to other less well understood surfacecharacteristics of the fibers which are generated by the hereindescribed procedure for modifying the fibers. This theory for theproposed mechanism is supported by the existence of filters capable ofhighly efficient leucocyte depletion of human red blood cell suspensionsand which have pore sizes as small as 0.5 μm, through which red cellspass freely and completely with no clogging, with applied pressure ofthe same magnitude as that used in the present invention.

On the other hand, the filters of the present invention, which typicallyhave pore diameters larger than about 0.5 μm, abruptly stop the flow ofred blood cells when the porous medium is contacted by the red cells.This suggests that the valve-like action is not related to or caused bypore size or by a filtration mechanism. The mechanism of this valve-likeaction is not well understood, but it may reflect zeta potential-relatedaggregation of the red cells as they reach the filter surface, forming abarrier which prevents or blocks further flow of a biological fluidcontaining red cells through the porous medium.

In one embodiment of the invention, the red cell filter assemblypreferably includes a preferred range for the fiber surface area ofabout 0.04 to about 0.3 M², and, more preferably, about 0.06 to about0.20 M². A preferred range for the porous medium flow area is about 3 toabout 8 cm², and a more preferred range is about 4 to about 6 cm². Apreferred range for the voids volume is about 71% to about 83%, and amore preferred range is from about 73% to about 80%. Because of itssmall size, a preferred device in accordance with this variation of theinvention typically exhibits a low hold-up volume. For example, when thebiological fluid processed is PRP, the device in accordance with thisvariation of the invention retains internally only about 0.5 to about 1cc of PRP, representing less than a 0.5% loss of platelets.

In another variation of the devices of this invention, the PRP derivedfrom a single unit of about 450 cc of human blood is passed, typicallyduring a flow interval of about 10 to 40 minutes, through a filtercomprising a porous medium, preferably comprising grafted fibers, with asurface area in the range of about 0.08 to about 1.0 square meters, andmore preferably about 0.1 to about 0.7 square meters, with a voidsvolume in the range of about 50% to about 89%, and more preferably about60% to about 85%. The filter element is preferably of right cylindricalform with the ratio of diameter to thickness preferably in the range ofabout 7:1 to about 40:1. The range of fiber diameter is preferred to beabout 1.0 to about 4 μm and is more preferred to be in the range ofabout 2 to about 3 μm. In relation to the previous variation of theinvention, this variation is made with higher fiber surface area, higherporous medium flow area, smaller porous medium density, and an increasedvoids volume.

All of these parameters can be varied; for example, the diameter of theporous medium could be reduced and the thickness increased whileretaining the same total quantity of fiber, or the fibers could belarger in diameter while increasing the total quantity of fiber, or thefibers could be packed as opposed to preformed into a cylindrical disc.Such variations fall within the purview of this invention.

Another variation of this invention may comprise a porous medium whereinthe upstream portion is of a higher density than the downstream portion.For example, the porous medium may comprise a higher density upstreamlayer for blocking the passage of red blood cells and a lower densitydownstream layer for the depletion of leucocytes.

In one embodiment of this invention, the fiber is surface modified inthe same manner as in the preceding versions, but the fiber surface areaelement is increased while, at the same time, the density is somewhatreduced. In this way, the automatic blockage of flow on contact by redcells is combined with very high efficiency of leucocyte depletion.

A preferred range of fiber surface area for this variation of theinvention is from about 0.3 to about 2.0 M², and a more preferred rangeis from about 0.35 to about 0.6 M². The upper limits of fiber surfacearea reflect the desire to accomplish the filtration in a relativelyshort time period, and may be increased if longer filtration times areacceptable. A preferred voids volume for the red cell barrier assemblyis in the range of about 71% to about 83%, and more preferably about 75%to about 80%. A preferred flow area is from about 2.5 to about 10 cm²,and a more preferred area is from about 3 to about 6 cm². Leucocytedepletion efficiencies in excess of about 99.9%, which corresponds to anaverage residual leucocyte content per unit of less than about 0.05×10⁶,can be obtained.

In a preferred embodiment of the invention, a porous medium for use witha biological fluid such as a supernatant layer (e.g., PRP) may comprisethe type of device disclosed in U.S. Pat. No. 4,880,548, hereinincorporated by reference. In a preferred embodiment of the invention, aporous medium for use with a biological fluid such as a sediment layer(e.g., PRC), may comprise the type of device disclosed in U.S. Pat. No.4,925,572 and U.S. Pat. No. 4,923,620, both incorporated herein byreference.

As noted above, as the sediment layer such as PRC is expressed from thecollection bag, it may be processed through a device having a leucocytedepletion element in order to reduce the leucocyte content of thesediment layer. In accordance with the invention, the porous medium forremoving leucocytes from the packed red cell component of a biologicalfluid comprises a leucocyte removal element or porous medium. Thepreferred element is typically made using radiation grafted melt blownfibers having an average diameter of from about 1 to about 4 μm,preferably from about 2 to about 3 μm. Polybutylene terephthalate (PBT)web, which is a preferred material, may be hot compressed to a voidsvolume of about 65% to about 90% and preferably to about 73% to about88.5%.

SEPARATION ASSEMBLY

The present invention involves the separation of one or more componentsfrom a biological fluid. In accordance with the present invention, abiological fluid, particularly blood, may be exposed to a separationmedium suitable for passing at least one component of the biologicalfluid, particularly plasma, therethrough, but not other components ofthe biological fluid, particularly platelets and/or red cells. Cloggingof the separation medium by these other components is minimized orprevented.

In the embodiment of the invention which includes a separation assembly14, preferably a non-centrifugal separation device, the supernatantlayer (e.g., PRP) may be passed through a leucocyte depletion assembly,and then passed through the non-centrifugal separation device 14, whereit may be processed and separated into components, which may beseparately collected in container 15 and container 16. In a preferredembodiment, if the supernatant fluid is PRP, it may be separated intoplasma and platelet concentrate as the PRP passes through thenon-centrifugal separation device.

As shown in FIG. 5, a preferred separation device of the presentinvention comprises a housing 210 having first and second portions 210a,220b joined in any convenient manner. For example, the first and secondhousing portions 210a, 210b may be joined by means of an adhesive, asolvent, or one or more connectors. The housing 210 also has an inlet211 and first and second outlets 212 and 213, respectively, such that afirst fluid flow path 214 is established between the inlet 211 and firstoutlet 212 and a second fluid flow path 215 is established between theinlet 211 and the second outlet 213. A separation medium 216 havingfirst and second surfaces 216a, 216b is positioned inside the housing210 between the first and second housing portions 210a, 210b. Further,the separation medium 216 is positioned parallel to the first fluid flowpath 214 and across the second fluid flow path 215.

Embodiments of the present invention may be configured in a variety ofways to ensure maximum contact of the biological fluid with the firstsurface 216a of separation medium 216 and to reduce or eliminateclogging on the first surface 216a of the separation medium. Forexample, the separation device may include a first shallow chamberfacing the first surface 216a of the separation medium 216. The firstchamber may include an arrangement of ribs which spread the flow ofbiological fluid over the entire first surface 216a of the separationmedium 216. Alternatively, the first chamber may include one or morechannels, grooves, conduits, passages, or the like which may beserpentine, parallel, curved, or a variety of other configurations.

The fluid flow channels may be of any suitable design and construction.For example, the channels may have a rectangular, triangular, orsemi-circular cross section and a constant depth. Preferably, thechannels have a rectangular cross section and vary in depth, forexample, between inlet 211 and outlet 212.

In the embodiment shown in FIGS. 6, 7, and 8, the inlet 211 of thehousing 210 is connected to serpentine fluid flow channels 220, 221, and222 which face the first surface 216a of the separation medium 216.These channels 220-222 separate the inlet flow of biological fluid intoseparate flow paths tangential to the first surface 216a of theseparation medium 216. Extending along the first surface 216a, theserpentine fluid flow channels 220, 221, and 222 may be recombined atfirst outlet 212 of the housing 210.

Embodiments of the present invention may also be configured in a varietyof ways to minimize back pressure across the separation medium 216 andto ensure a sufficiently high velocity of flow to the second outlet 213to prevent fouling of surface 216a, while minimizing hold-up volume. Theseparation device includes a second shallow chamber facing the secondsurface 216b of the separation medium 216. Like the first chamber, thesecond chamber may include an arrangement of ribs or may comprise one ormore channels, grooves, conduits, passages, or the like which may beserpentine, parallel, curved, or have a variety of other configurations.

The fluid flow channels may be of any suitable design and construction.For example, the channels may have a rectangular, semi-circular, ortriangular cross section and a constant or variable depth. In theembodiment shown in FIGS. 9-11, several serpentine fluid flow channels231, 232, 233, 234, and 235 face the second surface 216b of theseparation medium 216. Extending along the second surface 216b, theserpentine fluid flow channels 231-235 may be recombined at the secondoutlet 213.

Ribs, walls, or projections 241-245 may be used to define the channels220-222, 231-235 of the first and second chambers and/or may support orposition the separation medium 216 within the housing 210. In apreferred embodiment of the invention, there are more walls 242 in thesecond chamber than in the first chamber to prevent deformation of theseparation medium 216 caused by pressure differential through theseparation medium.

In use, a biological fluid, e.g., whole blood or PRP, is fed undersufficient pressure into the inlet 211 of housing 210 from any suitablesource of the biological fluid. For example, the biological fluid may beinjected from a syringe into the inlet 211 or it may be forced into theinlet 211 from a flexible bag using a gravity head, a pressure cuff, oran expressor. From the inlet 211, the biological fluid enters thechannels 220-222 of the first chamber and passes tangentially orparallel to the first surface 216a of the separation medium 216 on theway to the first outlet 212 via the first fluid flow path 214. At leastone component of the biological fluid, e.g., plasma, passes through theseparation medium 216, enters the channels 231-235 of the secondchamber, and is directed toward the second outlet 213 via the secondfluid flow path 215. As the biological fluid continues along the firstflow path 214 tangentially or parallel to the first surface 216a of theseparation medium 216, more and more plasma crosses the separationmedium 216. A plasma-depleted fluid then exits the housing 210 at thefirst outlet 212 and is recovered in one container 217 while plasmaexits the housing 210 at the second outlet 213 and is recovered inanother container 218.

While any biological fluid containing plasma may be used in conjunctionwith the present invention, the present invention is particularlywell-suited for use with blood and blood products, especially wholeblood or PRP. By subjecting PRP to processing in accordance with thepresent invention, PC and platelet-free plasma may be obtained withoutcentrifugation of the PRP and the attendant disadvantages discussedabove. Likewise, platelet-free plasma may be obtained from whole blood.The biological fluid may be supplied in any suitable quantity consistentwith the capacity of the overall device and by any suitable means, e.g.,in a batch operation by, for example, a blood bag connected to anexpressor or a syringe, or in a continuous operation as part of, forexample, an apheresis system. Exemplary sources of biological fluidinclude a syringe 219, as shown in FIG. 5, or a biological fluidcollection and processing system such as that disclosed in U.S. patentSer. No. 07/609,654, filed Nov. 6, 1990, incorporated herein byreference. A source of biological fluid may also include an apheresissystem, and/or may include a system in which biological fluid isrecirculated through the system.

The separation medium and housing may be of any suitable material andconfiguration and the separation medium may be arranged in the presentinventive device in any suitable manner so long as the biological fluidflow tangential or parallel to the separation medium is maintained to asufficient extent to avoid or minimize substantial platelet adhesion tothe separation membrane. The hydrodynamics of flow parallel to a surfaceare indeed believed to be such that during flow parallel to the surface,platelets develop a spin which causes them to be recovered from thesurface. While the preferred device has one inlet and two outlets, otherconfigurations can be employed without adversely affecting the properfunctioning of the device. For example, multiple inlets for a biologicalfluid may be used so long as the biological fluid flows tangentiallyacross the face of the separation medium. The plasma may preferably bestored in a region separated from the separation medium in order toavoid possible reverse flow of the plasma back across the separationmedium to the plasma-depleted fluid.

One skilled in the art will recognize that platelet adhesion may becontrolled or affected by manipulating any of a number of factors:velocity of the fluid flow, configuration of the channel, depth of thechannel, varying the depth of the channel, the surface characteristicsof the separation medium, the smoothness of the medium's surface, and/orthe angle at which the fluid flow crosses the face of the separationmedium, among other factors. For example, the velocity of the firstfluid flow is preferably sufficient to remove platelets from the surfaceof the separation medium. Without intending to be limited thereby, avelocity in excess of about 30 cm/second has been shown to be adequate.

The velocity of the fluid flow may also be affected by the volume of thebiological fluid, by varying the channel depth, and by the channelwidth. For example, the channel depth may be varied from about 0.25 inchin the region 223 near the inlet 211 to about 0.001 inch, in the region224 near the outlet 212 as shown in FIG. 7. One skilled in the art willrecognize that a desired velocity may be achieved by manipulating theseand other elements. Also, platelets may not adhere as readily to aseparation medium having a smooth surface as compared to a membranehaving a rougher surface.

In accordance with the invention, the separation medium comprises aporous medium suitable for passing plasma therethrough. The separationmedium, as used herein, may include but is not limited to polymericfibers (including hollow fibers), polymeric fiber matrices, polymericmembranes, and solid porous media. Separation media according to theinvention remove plasma from a biological solution containing platelets,typically whole blood or PRP, without removing proteinaceous bloodcomponents and without allowing a substantial amount of platelets topass therethrough.

A separation medium, in accordance with the invention, preferablyexhibits an average pore rating generally or intrinsically smaller thanthe average size of platelets, and, preferably, platelets do not adhereto the surface of the separation medium, thus reducing pore blockage.The separation medium should also have a low affinity for proteinaceouscomponents in the biological fluid such as PRP. This enhances thelikelihood that the platelet-poor solution, e.g., platelet-free plasmawill exhibit a normal concentration of proteinaceous clotting factors,growth factors, and other needed components.

For the separation of about one unit of whole blood, a typicalseparation device according to the invention may include an effectivepore size smaller than platelets on the average, typically less thanabout 4 micrometers, preferably less than about 2 micrometers. Thepermeability and size of the separation device is preferably sufficientto produce about 160 cc to about 240 cc of plasma at reasonablepressures (e.g., less than about 20 psi) in a reasonable amount of time(e.g., less than about one hour). In accordance with the invention, allof these typical parameters may be varied to achieve a desired result,i.e., varied preferably to minimize platelet loss and to maximizeplatelet-free plasma production.

In accordance with the invention, a separation medium formed of fibersmay be continuous, staple, or melt-blown. The fibers may be made fromany material compatible with a biological fluid containing platelets,e.g., whole blood or PRP, and may be treated in a variety of ways tomake the medium more effective. Also, the fibers may be bonded, fused,or otherwise fixed to one another, or they may simply be mechanicallyentwined. A separation medium formed of a membrane, as the term is usedherein, refers to one or more porous polymeric sheets, such as a wovenor non-woven web of fibers, with or without a flexible porous substrate,or may comprise a membrane formed from a polymer solution in a solventby precipitation of a polymer when the polymer solution is contacted bya solvent in which the polymer is not soluble. The porous, polymericsheet will typically have a substantially uniform, continuous matrixstructure containing a myriad of small largely interconnected pores.

The separation medium of this invention may be formed, for example, fromany synthetic polymer capable of forming fibers or a membrane. While notnecessary to the apparatus or method of the invention, in a preferredembodiment the polymer is capable of serving as a substrate for graftingwith ethylenically unsaturated monomeric materials. Preferably, thepolymer should be capable of reacting with at least one ethylenicallyunsaturated monomer under the influence of ionizing radiation or otheractivation means without the matrix being adversely affected. Suitablepolymers for use as the substrate include, but are not limited to,polyolefins, polyesters, polyamides, polysulfones, polyarylene oxidesand sulfides, and polymers and copolymers made from halogenated olefinsand unsaturated nitriles. Preferred polymers are polyolefins,polyesters, and polyamides, e.g., polybutylene terephthalate (PBT) andnylon. In a preferred embodiment, a polymeric membrane may be formedfrom a fluorinated polymer such as polyvinylidene difluoride (PVDF). Themost preferred separation media are a microporous polyamide membrane ora polycarbonate membrane.

Surface characteristics of a fiber or membrane can be affected as notedabove for a porous medium. An exemplary radiation grafting techniqueemploys at least one of a variety of monomers each comprising anethylene or acrylic moiety and a second group, which can be selectedfrom hydrophilic groups (e.g., --COOH, or --OH) or hydrophobic groups(e.g., a methyl group or saturated chains such as --CH₂ CH₂ CH₃)Grafting of the fiber or membrane surface may also be accomplished bycompounds containing an ethylenically unsaturated group, such as anacrylic moiety, combined with a hydroxyl group, such as, hydroxyethylmethacrylate (HEMA). Use of HEMA as the monomer contributes to a veryhigh CWST. Analogues with similar characteristics may also be used tomodify the surface characteristics of fibers.

In accordance with an embodiment of the invention, the separating mediummay be surface-modified, typically by radiation grafting, in order toachieve the desired performance characteristics, whereby platelets areconcentrated with a minimum of medium blocking, and whereby theresulting plasma solution contains essentially all of its nativeproteinaceous constituents. Exemplary membranes having a low affinityfor proteinaceous substances are disclosed in U.S. Pat. Nos. 4,886,836;4,906,374; 4,964,989; and 4,968,533, all incorporated herein byreference.

Suitable membranes in accordance with an embodiment of the invention maybe microporous membranes and may be produced by a solution castingmethod.

As noted above, establishing a tangential flow of the biological fluidbeing processed parallel with or tangential to the face of theseparation medium minimizes platelet collection within or passagethrough the separation medium. In accordance with the invention, thetangential flow can be induced by any mechanical configuration of theflow path which induces a high local fluid velocity at the immediatemembrane surface. The pressure driving the biological fluid across theseparation medium may be derived by any suitable means, for example, bygravity head or by an expressor.

The tangential flow of the biological fluid may be directed tangentialor parallel to the face of the separation medium in any suitable manner,preferably utilizing a substantial portion of the separation mediumsurface while maintaining a sufficient flow to ensure that the plateletsdo not clog or block the pores of the separation medium. The flow of thebiological fluid is preferably directed tangentially or parallel to theface of the separation medium through use of at least one serpentinefluid flow channel which is designed to maximize utilization of theseparation medium, ensure a sufficiently total area contact between thebiological fluid and the separation medium, and maintain a sufficientflow of biological fluid to minimize or prevent platelet adhesion to theseparation medium. Most preferably, several (e.g., three or more) fluidflow channels are utilized so as to fix the separation medium in placeand to prevent sagging of the membrane due to the applied pressure. Thefluid flow channels may be of any suitable design and construction andpreferably are variable with respect to depth such as depth to maintainoptimal pressure and fluid flow across the face of the separationmedium. Fluid flow channels may also be utilized on the side of theseparation medium opposite the biological fluid tangential flow tocontrol the flow rate and pressure drop of a platelet-poor fluid, suchas plasma.

The present inventive device may similarly be part of an apheresissystem. The biological fluid to be processed, the platelet-richsolution, and/or the platelet-poor solution may be handled in either abatch or continuous manner. The sizes, nature, and configuration of thepresent inventive device can be adjusted to vary the capacity of thedevice to suit its intended environment.

GAS INLET/OUTLET

Under certain circumstances, it may be desirable to maximize therecovery of a biological fluid retained or entrapped in various elementsof the biological fluid processing system. For example, under typicalconditions, using a typical device, the biological fluid will drainthrough the system until the flow is stopped, leaving some of the fluidin the system. In one embodiment of the invention, the retained fluidmay be recovered by using at least one gas inlet and/or at least one gasoutlet. An exemplary configuration of this embodiment is shown in FIG.3.

The gas outlet is a porous medium which allows gas that may be presentin a biological fluid processing system when the biological fluid isprocessed in the system, out of the system. The gas inlet is a porousmedium which allows gas into a biological fluid processing system.

As used herein, gas refers to any gaseous fluid, such as air, sterilizedair, oxygen, carbon dioxide, and the like; it is intended that theinvention is not to be limited to the type of gas used.

The gas inlet and gas outlet are chosen so that the sterility of thesystem is not compromised. The gas inlet and the gas outlet areparticularly suited for use in closed systems, or may be used later, forexample, within about 24 hours of a system being opened.

The gas inlet and the gas outlet each comprise at least one porousmedium designed to allow gas to pass therethrough. A variety ofmaterials may be used, provided the requisite properties of theparticular porous medium are achieved. These include the necessarystrength to handle the differential pressures encountered in use and theability to provide the desired filtration capability while providing thedesired permeability without the application of excessive pressure. In asterile system, the porous medium should also preferably have a porerating of about 0.2 micrometer or less to preclude bacteria passage.

The gas inlet and gas outlet may comprise a porous medium, for example,a porous fibrous medium, such as a depth filter, or a porous membrane orsheet. Multilayered porous media may be used, for example, amultilayered microporous membrane with one layer being liquophobic andthe other liquophilic.

Preferred starting materials are synthetic polymers includingpolyamides, polyesters, polyolefins, particularly polypropylene andpolymethylpentene, perfluorinated polyolefins, such aspolytetrafluoroethylene, polysulfones, polyvinylidene difluoride,polyacrylonitrile and the like, and compatible mixtures of polymers. Themost preferred polymer is polyvinylidene difluoride. Within the class ofpolyamides, the preferred polymers include polyhexamethylene adipamide,poly-Δ-caprolactam, polymethylene sebacamide, poly-7-aminoheptanoamide,polytetramethylene adipamide (nylon 46), or polyhexamethyleneazeleamide, with polyhexamethylene adipamide (nylon 66) being mostpreferred. Particularly preferred are skinless, substantiallyalcohol-insoluble, hydrophilic polyamide membranes, such as thosedescribed in U.S. Pat. No. 4,340,479.

Other starting materials may also be used to form the porous media ofthis invention including cellulosic derivatives, such as celluloseacetate, cellulose propionate, cellulose acetate-propionate, celluloseacetate-butyrate, and cellulose butyrate. Non-resinous materials, suchas glass fibers, may also be used.

The rate of air flow through the gas outlet or the gas inlet can betailored to the specific biological fluid or fluids of interest. Therate of air flow varies directly with the area of the porous medium andthe applied pressure. Generally, the area of the porous medium isdesigned to enable the biological fluid processing system to be primedin a required time under the conditions of use. For example, in medicalapplications it is desirable to be able to prime an intravenous set infrom about 30 to about 60 seconds. In such applications as well as inother medical applications, the typical porous medium is a membrane,which may be in the form of a disc which has a diameter from about 1 mmto about 100 mm, preferably from about 2 mm to about 80 mm, and morepreferably from about 3 mm to about 25 mm.

In accordance with the invention, the processing system may be providedwith a gas inlet to permit the introduction of gas into the system,and/or with a gas outlet to permit gases in the various elements of thesystem to be separated from the biological fluid to be processed. Thegas inlet and the gas outlet may be used together in connection with atleast one assembly, porous medium, or container in the system, or theymay be used separately.

To that end, a gas inlet or gas outlet may be included in any of thevarious elements of the biological fluid processing system. By way ofillustration, a gas inlet or gas outlet may be included in at least oneof the conduits which connect the different containers, in a wall of thecontainers that receive the processed biological fluid, or in a port onor in one of those containers. The gas inlet or gas outlet may also beincluded on or in a combination of the elements mentioned above. Also,an assembly or porous medium may include one or more gas inlet or gasoutlet as described above. Generally, however, it is preferred toinclude a gas inlet or gas outlet in the conduits which connect thecontainers or in the functional medical device. Included within thescope of the invention is the use of more than one gas inlet or gasoutlet in any conduit, receiving container, assembly, or porous medium.

It will be apparent to one skilled in the art that the placement of agas inlet or a gas outlet may be optimized to achieve a desired result.For example, it may be desirable to locate the gas inlet upstream of aporous medium and in or as close to the first container as is practicalin order to maximize the recovery of biological fluid. Also, it may bedesirable to locate the gas outlet downstream of the porous medium andas close to the receiving container as is possible in order to maximizethe volume of gas that is removed from the system.

Such placement of the gas inlet or gas outlet is particularly desirablewhere there is only one gas inlet or gas outlet in the system.

In accordance with the invention, recovery from the various elements ofthe biological fluid processing system may be maximized. For example,whole blood is subjected to a processing step, resulting in separate PRPand PRC layers. Then, the separate fractions of blood components areexpressed to their respective receiving containers through theappropriate conduits and porous media, if any. Blood product that hasbecome entrapped in these elements during processing may be recoveredeither by passing purge gas through the conduits and porous media, or bycreating at least a partial vacuum in the system to draw out theretained blood product and to permit it to drain into the appropriatereceiving container or assembly.

The purge gas may be from any of a number of sources. For example, thebiological fluid processing system may be provided with a storagecontainer for the storage of the purge gas, the purge gas may be the gasthat was removed from the system during the processing function, or thepurge gas may be injected aseptically into the system from an outsidesource (e.g., through a syringe). For example, it may be desirable touse sterile purge gas that has been sterilized in a separate containerapart from the biological fluid processing system.

In accordance with the invention, a clamp, closure, or the like may bepositioned on or in any or all of the conduits in order to facilitate adesired function, i.e., establishing a desired flow path for biologicalfluid or gas. For example, when processing a biological fluid (e.g.,PRP) through a system such as is illustrated in FIG. 3, during theremoval of gases from the conduits and the leucocyte depletion assembly,it may be desirable to clamp the conduit immediately below gas outlet54. When it is desirable to use the gas inlet 53 to maximize therecovery of a biological fluid, the clamp below gas outlet 54 isreleased, and a clamp in the conduit adjacent to gas intake 53 isopened. As exemplified in FIG. 3, other gas inlets and gas outlets(e.g., 51 and 52) may be operated in a similar manner.

With continued reference to FIG. 3, as a column of biological fluid(e.g., PRP) flows from the first container 11 through the conduit andthe leucocyte depletion assembly 13, towards a satellite bag (notshown), it drives the gas in those elements towards the gas outlet 54.

The gas outlet may comprise a branching element with three legs. One legmay include a liquophobic porous medium which preferably has a pore sizeof not greater than 0.2μ. At the branching element, gas ahead of thecolumn of biological fluid moves into one leg of the branching element.Since the gas passes through the liquophobic porous medium, but thebiological fluid does not, the gas is separated from the PRP and isprecluded from entering a satellite bag (not shown).

The gases separated by the gas outlet 54 may be vented from the system,or they may be collected in a gas container (not shown) and returned tothe system as a purge gas to facilitate the recovery of biological fluidthat becomes trapped in the various components of the system.

After the system is primed and the gas outlet is inactivated, the clampadjacent to the containers or assembly is opened to allow the containersto fill with processed biological fluid. This continues until thecollection bag 11 collapses. In order to recover the very valuablebiological fluid retained in the system, ambient air or a sterile gasmay enter the system through gas inlet 51 or 53. If gas inlet 51 or 53is a manual inlet means, a closure is opened or a clamp released; if thegas inlet 51 or 53 is automatic, the pressure differential between thegas inlet and the containers will cause the air or gas to flow throughthe conduits, through the porous media, and towards the respectivecontainers. In the process, retained biological fluid that is trapped inthose elements during processing are recovered from those components andcollected in the containers. It should be noted that the purge air orgas is preferably separated from the biological fluid at gas outlet 52or 54, so that little, if any, purge gas will be received by thecontainers. This may be accomplished by clamping the conduit downstreamof the gas outlet 52 or 54. In another embodiment of the invention, thepurge air or gas may be separated from the system through a gas outletlocated in the bag itself.

BRACKET

In another embodiment, the invention also includes a bracket whichsecures a filter assembly comprising a porous medium or one or morecomponents of an assembly in place during centrifugation so that it(they) is (are) not damaged by the stresses generated duringcentrifugation.

The blood collection and processing assembly 10, with one or moresatellite bags attached or connected via a conduit, may be usedintegrally to separate components from whole blood. This embodiment ofthe invention will be better understood by reference to the exemplaryconfiguration shown in FIG. 4. During the centrifugation step in whichthe red cells are concentrated at the bottom of the collection bag,forces of up to about 5000 times gravity (5000 G) or more may begenerated. Therefore, the collection bag is preferably flexible, as arethe other bags, allowing them to settle to the bottom and against thewalls of a centrifuge bucket 120, so that the bags themselves aresubject to little or no stress.

In contrast to the flexibility and pliability of the bags and tubing,the porous medium is typically contained in a rigid plastic housing (thecombination of which is termed a filter assembly). The PRC housing istypically of larger dimensions than the PRP housing, and is thereforesubject to an increased probability of suffering or causing damageduring centrifugation. For example, a typical PRC filter assembly mayweigh about 20 grams (about 0.04 lbs), but its effective weight may be5000 times greater, or about 200 lbs, under centrifugation conditions of5000 G. In conventional centrifugation systems it is therefore difficultto avoid shattering the plastic housing. Even careful placement of thePRC filter assembly in the centrifuge bucket is likely to result indamage to the plastic tubing or to the bags. Furthermore, it isundesirable to enlarge the centrifuge bucket to accommodate the filterassembly in the bucket during the centrifugation step, as this would notonly require the use of a larger and more costly centrifuge, but itwould also require retraining the thousands of blood processingtechnicians to expertly assemble the blood bag sets into a new type ofcentrifuge bucket.

Accordingly, it is desirable that an improved blood collection andprocessing system or set should be usable with existing centrifugebuckets. In accordance with the invention, this is preferablyaccomplished by locating the PRC filter assembly away from the greatestamount of G force; this is more preferably outside or partly outside ofthe conventionally used centrifuge bucket, in the manner shown in FIG.4.

In FIG. 4 the bucket 120 depicts a centrifuge bucket such as is used incurrent blood bank practice. These buckets are typically constructedwith heavy walls of high strength steel which enclose open space 121into which the blood bag, its satellite bags, and the interposed tubingmay be placed. The bracket 122 used to hold a filter assembly may bemade of any high strength material, preferably metal or metallic alloy;titanium or stainless steel are more preferred for their strength andthe ease with which sanitary conditions can be maintained. The lowerportion 123 of bracket 122 is configured to cooperatively fit intocavity 121, preferably at a depth of about 0.5 to about 1 cm. Springclips or other means may be used to position and/or retain bracket 122in the bucket 120. Groove 124 located in the upper portion of bracket122, is preferably configured to cooperatively accept the outlet port125 of the filter assembly 114, and to allow the bottom portion of thefilter assembly 114 to rest on the flat upper surfaces of bracket 122adjacent to groove 124. The central portion 126 of groove 124 may beproportioned such that port 125 of the filter assembly 114 fits into atleast a portion of the groove 124 with a friction fit. The ends ofgroove 124 are preferably reduced to a width such that flexible tubing112 connected to the inlet and outlet of the filter assembly 114 isfirmly retained, thereby helping to stabilize the filter assembly 114when positioned onto bracket 122. The unsupported portions of flexibletubing 112 then drop into the bucket in communication with the balanceof the blood collection set contained therein. It is preferred that thebracket 122 retain the filter assembly 114 so that the plane of theporous medium is substantially perpendicular to the G force createdduring operation of the centrifuge. Also, the bracket and filterassembly should be positioned on or in the centrifuge bucket withoutinterfering with the normal free-swinging action of the bucket 120 inthe centrifuge during rotation.

Because the PRP filter is typically relatively small and very light, itmay be positioned within the bucket with the bags and the tubing. Inanother embodiment of the invention, however, the groove 124 may beconfigured to hold more than one filter assembly, for example, both aPRC and a PRP filter assembly.

In another embodiment of the invention, a larger bracket may be employedto hold a first filter assembly and a second bracket holding a secondfilter assembly may be nested on top of the first bracket and filterassembly. One skilled in the art will recognize that various designs,configurations, and/or means may be employed to accomplish thesefunctions.

Another feature of the invention is the location and manner in which theporous medium, particularly the PRC medium, is mounted on the centrifugebucket during the centrifugation operation. Trials of a number of testfilter housings designed to fit within the centrifuge bucketconvincingly demonstrated that perforation of tubing lines by thehousing is a frequent occurrence during centrifugation. Also, it is verydifficult to design a housing that can reliably withstand d withoutshattering. Further, the existing centrifuge buckets are designed tocarry the conventional blood collection sets, which incorporate nofilter elements. Fitting the added bulk of a PRC filter assembly into aconventional bucket was, thus, very difficult. These very seriousproblems were eliminated by mounting the PRC filter assembly on abracket outside of the bucket. This provides adequate support for thefilter assembly by cooperatively arranging flange portion 127 of bracket122 (FIG. 4) to accommodate the contours of the centrifuge bucket.Furthermore, bracket 122 is preferably positioned above the top of thebucket, a location which is much closer to the center of rotation of thecentrifuge that the force to which the filter assembly is subjected isabout 40% to about 60% that of the bottom of the bucket 120.Additionally, the narrow slots at each end of the bracket hold thetubing connections firmly, and permit the tubes to drop back into thebowl. Surprisingly, the suspended portions of the tubing tolerate thecentrifuging operation very well.

A system according to the present invention may be used in conjunctionwith other assemblies or porous media, including filtration and/orseparation devices, e.g., a device for removing leucocytes from aplatelet-containing solution or concentrate. Exemplary devices aredisclosed in U.S. Pat. No. 4,880,548, and U.S. Pat. No. 4,925,572incorporated herein by reference in their entirety.

Housings can be fabricated from any suitably impervious material,including an impervious thermoplastic material. For example, the housingmay preferably be fabricated by injection molding from a transparent ortranslucent polymer, such as an acrylic, polystyrene, or polycarbonateresin. Not only is such a housing easily and economically fabricated,but it also allows observation of the passage of the fluid through thehousing.

The housing into which the porous medium is sealed or interference fitis designed to achieve convenience of use, rapid priming, and efficientair clearance.

While the housing may be fashioned in a variety of configurations, thehousing of a porous medium according to the present invention preferablycomprises a housing such as that disclosed in U.S. Pat. Nos. 4,880,548;4,923,620; and 4,925,572, which are generally similar in configurationto housing 114 in FIG. 4.

A number of additional containers may be in communication with thebiological fluid processing system, and can be utilized to definedifferent flow paths. For example, an additional satellite bagcontaining physiological solution may be placed in communication withthe biological fluid processing system upstream of the leucocytedepletion assembly (e.g., through the gas inlet), and the solution maybe passed through the leucocyte depletion assembly so that thebiological fluid that was held up in the assembly can be collected.

Similarly, a satellite bag containing physiological solution may beplaced in communication with the biological fluid processing systemdownstream of the leucocyte depletion assembly (e.g., through the gasoutlet), and the solution may be passed through the leucocyte depletionassembly so that the biological fluid that was held up in the assemblycan be later collected.

It will be appreciated that when the biological fluid from thecollection bag 11 is expressed towards the containers, some of thebiological fluid may be trapped in the conduits and/or the porousmediums. For example, 8 cc to 35 cc is typically retained in the system;but as little as 2cc to as much as 150 cc or more may be retained insome types of systems.

In an embodiment of the invention (not shown), air or gas may be storedin at least one gas container; upon opening of valve or clamp means inthe conduits, gas can be fed through them to purge the conduits andassemblies, thereby facilitating the recovery of biological fluid thatmay have been trapped during processing.

Preferably, the purge air or gas is fed to the conduits at a point asclose as is reasonably possible to container 11 to maximize the volumeof biological fluid recovered. The air or gas container is preferablyflexible so that the gas therein may be fed to the system merely bysimple compression. The biological fluid containers and the air or gascontainers may be composed of the same material.

Priming, as used herein, refers to wetting or priming the inner surfacesof a device or assembly prior to its actual use allowing a separateassembly to be injected into the system. A valve or clamp is opened toallow fluid to flow through the assembly; then, with the passage offluid through the assembly, gas downstream of the fluid is expelledthrough the gas outlet until fluid reaches a branching element, at whichpoint the clamp is closed. With the clamp in a closed position, theconnector downstream of the gas outlet may be opened or readied for usewithout fluid in the assembly dripping through the connector.

In accordance with the invention, the biological fluid collection andprocessing assembly should be able to withstand rigorous sterilizationand centrifugation environments, typically consisting of radiationsterilization (at about 2.5 megarads), and/or autoclaving (at about 110°C. to about 120° C. for about 15 to 60 minutes), and/or centrifugation(typically about 2500 to 3500 G for about 5 to 15 minutes; however,depending on which biological fluid component is intended to havemaximum recovery, the centrifugation may be about 5000 G for about 10 to20 minutes).

The invention also involves a method for collecting and processing bloodcomprising collecting whole blood in a container; centrifuging the wholeblood; passing the supernatant layer of the centrifuged blood through afirst porous medium, the first porous medium comprising at least one ofa leucocyte depletion medium, a red cell barrier medium, and a combinedleucocyte depletion red cell barrier medium; and passing the sedimentlayer of the centrifuged blood through a second porous medium, thesecond porous medium comprising a leucocyte depletion medium.

This invention may also include a method for processing a biologicalfluid comprising passing a biological fluid from a first container to afirst porous medium comprising a red cell barrier medium, wherein thebiological fluid passes in a first flow path; and passing a biologicalfluid from a first container to a second porous medium comprising aleucocyte depletion medium, wherein the biological fluid passes in asecond flow path.

In general, donated whole blood is processed as soon as practicable inorder to more effectively reduce or eliminate contaminating factors,including but not limited to leucocytes and microaggregates.

Leucocyte depletion has been heretofore typically performed duringtransfusion at bedside, however, in accordance with the subjectinvention leucocyte depletion is accomplished during the initialprocessing of the whole blood, which in United States practice isgenerally within 8 hours of collection from the donor. After the redcells have been sedimented by centrifuging, the supernatant PRP isexpressed from the blood collection bag into a first satellite bagthrough one or more porous media which diminish the amount of leucocytesand/or block red cells, and the PRC remaining in the blood collectionbag is then passed through a porous medium which removes leucocytes intoa second satellite bag.

In general, using the Figures for reference, the biological fluid (e.g.,donor's whole blood) is received directly into the collection bag 11.The collection bag 11, with or without the other elements of the system,may then be centrifuged in order to separate the biological fluid into asupernatant layer 31 and a sediment layer 32. After centrifugation, ifwhole blood is used, the supernatant layer is primarily PRP, and thesediment layer is primarily PRC. The biological fluid may be expressedfrom the collection bag as separate supernatant and sediment layers,respectively. There may be a clamp or the like between the collectionbag 11 and the flexible tubing 25, or within the tubing, to prevent theflow of the supernatant layer from entering the wrong conduit.

Movement of the biological fluid through the system is effected bymaintaining a pressure differential between the collection bag and thedestination of the biological fluid (e.g., a container such as asatellite bag or a needle on the end of a conduit). The system of theinvention is suitable for use with conventional devices for establishingthe pressure differential, e.g., an expressor. Exemplary means ofestablishing this pressure differential may be by gravity head, applyingpressure to the collection bag (e.g., by hand or with a pressure cuff),or by placing the other container (e.g., satellite bag) in a chamber(e.g., a vacuum chamber) which establishes a pressure differentialbetween the collection bag and the other container. Also included withinthe scope of the invention may be expressors which generatesubstantially equal pressure over the entire collection bag.

As the biological fluid passes from one bag to the next, it may passthrough at least one porous medium. Typically, if the biological fluidis the supernatant layer (e.g., PRP), it may pass from the collectionbag through one or more devices or assemblies comprising one or moreporous media--a leucocyte-depletion medium, a red cell barrier medium, aporous medium which combines the red cell barrier with leucocytedepletion in one porous medium, or a leucocyte depletion medium and ared cell barrier medium in series. The supernatant layer 31 is expressedfrom the first container 11 until flow is stopped, typically by closinga clamp in conduit 20, or automatically if the assembly includes a redcell barrier medium 12. Preferably, the supernatant layer passes througha red cell barrier medium and then through a leucocyte depletion medium.The supernatant layer is then leucocyte-depleted after passing throughthe leucocyte depletion medium. Additional processing, if desired, mayoccur downstream of the leucocyte depletion medium, either connected tothe system or after being separated from the system.

The sediment layer 32 in collection bag 11 may be passed through aleucocyte depletion assembly 17 and into a container 18, such as asatellite bag. Typically, the collection bag 11, now containingprimarily red cells, is then subjected to a pressure differential, asnoted above, in order to prime the leucocyte depletion assembly 17 andto initiate flow.

In accordance with an additional embodiment of the invention, a methodis provided whereby the recovery of various biological fluids trapped orretained in various elements of the system is maximized, either bycausing a volume of gas behind the trapped or retained biological fluidto push the fluid through those elements and into the designatedcontainer, assembly, or porous medium, or by drawing the trapped orretained fluid into the designated container, assembly, or porous mediumby pressure differential (e.g., gravity head, pressure cuff, suction,and the like). This provides for a more complete emptying of thecontainer, assembly, or porous medium. Once the container is emptiedcompletely, the flow is stopped automatically.

In order that the invention herein described may be more fullyunderstood, the following examples are set out regarding use of thepresent invention. These examples are for illustrative purposes only andare not to be construed as limiting the present invention in any manner.

EXAMPLES EXAMPLE 1

The biological fluid processing system used to perform the first exampleincludes a blood collection bag, separate PRP and PRC leucocytedepletion assemblies, as well as separate PRP and PRC satellite bags. Inaddition, a red cell barrier medium between the collection bag and thePRP leucocyte depletion assembly precludes the flow of red cells intothe PRP satellite bag.

The red cell barrier assembly includes a porous medium for blocking flowupon contact by red cells when the PRP passes from the collecting bag.The red cell barrier assembly was preformed from 2.6 μm average diameterPBT fibers, which had been surface modified in accordance with theprocedures disclosed in U.S. Pat. No. 4,880,548, using hydroxyethylmethacrylate and methacrylic acid in a monomer ratio of 0.35:1 to obtaina CWST of 95 dynes/cm and a zeta potential of -11.4 millivolts. Theporous element's effective diameter was 2.31 cm, presenting a filterarea of 4.2 cm², thickness was 0.051 cm, voids volume was 75% (density,0.34 g/cc), and fiber surface area was 0.08m².

The volume of PRP held up within the housing was <0.4 cc, representing aloss of PRP due to hold-up of less than 0.2%. The flow stopped abruptlyas red cells reached the upstream surface of the red cell barrierassembly, and there was no visible evidence of red cells or hemoglobindownstream.

The PRP leucocyte depletion assembly containing a porous medium fordepleting leucocytes from PRP after the PRP passed through the red cellbarrier assembly, is described in U.S. Pat. No. 4,880,548. Similarly,the PRC leucocyte depletion assembly, containing a porous medium fordepleting leucocytes from the unit of PRC is described in U.S. Pat. No.4,925,572. The Example used a single unit of whole blood donated by avolunteer. The unit of blood was collected in a collection bag which waspre-filled with 63 ml of CPDA anti-coagulant. The collected blood wassubjected to soft-spin centrifugation in accordance with customary bloodbank practices. The collection bag was transferred, with care to avoiddisturbing its contents, to a plasma extractor, which was spring biasedto develop a pressure of about 90 mm Hg.

The pressure from the expressor drives the PRP from the collection bagthrough the red cell barrier assembly, the PRP filter assembly (where itis leucocyte depleted), and then to the satellite bag. As the PRP exitedthe collection bag, the interface between the PRC and PRP rose. When thered cells (present in the leading edge of the PRC layer), contacted thered cell barrier assembly, the flow was terminated, automatically, andwithout monitoring.

The PRC remaining in the collection bag is also processed. Thecollection bag is suspended and then squeezed to prime the PRC leucocytedepletion filter, and the PRC is leucocyte depleted. When the suspendedcollection bag is empty, the process stops automatically. The nowleucocyte depleted red cell product is eventually collected in thesatellite bag, and is available for transfusion into a patient asrequired.

The PRP previously collected in the satellite bag was then processedusing normal blood bank procedures (i.e., hard-spin centrifugation) toproduce PC and plasma.

EXAMPLE 2

Whole blood was collected into an Adsol™ donor set and was processedunder standard conditions to yield a unit of PRP. The PRP was thenfiltered to remove leucocytes using a filter device described in U.S.Pat. No. 4,880,548. The removal efficiency was >99.9%.

The filtered PRP unit was then placed in a pressure cuff to which apressure of 300 mm Hg was applied. The tubing exiting the bag (clampedclosed at this point) was connected to the inlet port of a separationdevice as shown in FIGS. 5, 6, and 9. A microporous polyamide membranehaving a pore rating of 0.65 microns was used as the separation mediumin the device. The area of the membrane was about 17.4 squarecentimeters. The depth of the first fluid flow path channels decreasedfrom about 0.03 cm near the inlet to about 0.01 cm near the outlet. Thedepth of the second fluid flow path channels was about 0.025 cm. Theoutlet ports of the device were connected to tubing which allowed thevolume of fluid exiting the device to be measured and saved foranalysis.

The test of the present invention was started by opening the clamp andallowing PRP to enter the device. Clear fluid (plasma) was observed toexit one port, and turbid fluid (platelet concentrate) exited the port.The duration of the test was 42 minutes in which 154 ml of plasma and 32ml of platelet concentrate was collected. The concentration of plateletsin the plasma was found to be 1.2×10⁴ /μl, while the concentration ofplatelets in the platelet concentration was found to be 1.43×10⁶ /μl.

The above results indicate that platelets can be concentrated to auseful level and plasma can be recovered in a reasonable time by adevice according to the invention.

EXAMPLE 3

Whole blood was collected and processed into blood components as inexample 2, and compared to that produced by conventional processing. Theresults comparing the blood component volumes to their respectiveleucocyte counts are listed in Table I, which shows the increasedefficiency of leucocyte removal over the conventional procedures. TableI also reflects the increased yield of plasma and correspondingdecreased yield of PRC resulting from the use of this invention.

                  TABLE I                                                         ______________________________________                                                      Conventional                                                                           Invention                                              ______________________________________                                        Whole Blood     450-500    450-500                                            volume (cc)                                                                   WBC-whole blood 2 × 10.sup.9                                                                       2 × 10.sup.9                                 PC volume (cc)  50-65      50-65                                              WBC-PC          ≈1 × 10.sup.8                                                              ≈1 × 10.sup.5                        Plasma volume (cc)                                                                            165-215    170-220                                            WBC-Plasma      <10.sup.8  <10.sup.8                                          PRC Volume (cc)*                                                                              335        320                                                WBC-PRC*        2 × 10.sup.9                                                                       1 × 10.sup.6                                 ______________________________________                                         *w/Adsol                                                                 

EXAMPLES 4-8

The blood collection sets used to perform the examples were in generalconformance with FIG. 1, without the optional red cell barrier mediumassembly, and the procedure was as described earlier, using an apparatusin accordance with FIG. 4 for the first centrifuging step.

The porous medium for depleting leucocytes from PRP was preformed into acylindrical filter element 2.5 cm in diameter and 0.33 cm thick, usingPBT fibers 2.6 μm in average diameter and of density 1.38 g/cc, whichhad been surface modified in accordance with the procedures disclosed inU.S. Pat. No. 4,880,548, using a mixture of hydroxyethyl methacrylateand methacrylic acid monomers in a weight ratio of 0.3:1. The porousmedium had a CWST of 95 dynes/cm and a zeta potential of -11.4millivolts at the pH of plasma (7.3). Fiber surface area was 0.52 M² andvoids volume was 80%.

The porous medium for depleting leucocytes from PRC, in accordance withequations (3) and (4) above, was calculated to obtain a leucocytedepletion efficiency better than log 3 (i.e. >99.9% reduction ofleucocyte content). This was accomplished by using 3.4 grams of 2.6 μmdiameter PBT fiber, about 13% more fiber than called for by equations(3) and (4), and, in order to further increase the leucocyte depletionefficiency, the voids volume was decreased to 81%. These changes boostedthe efficiency to better than log 4 (i.e., to >99.99%). When the PRC wasexpressed through this filter medium contained in a housing 6.4 cm indiameter, flow times in the desired 10 to 40 minute range were obtainedat 90 mm Hg applied pressure. The fiber surfaces had been modified inthe manner disclosed in U.S. Pat. No. 4,925,572, using a mixture ofhydroxyethyl methacrylate and methyl methacrylate in a weight ratio of0.27 to 1; the porous medium had a CWST of 66 dynes/cm.

The PRC porous medium described above was preceded by a pre-filterconsisting of five layers of PBT melt blown web laid up in the ordernoted below to a total thickness of 0.25 cm:

    ______________________________________                                                               Fiber                                                            Weight,      Diameter,                                              Grade     mg/cm.sup.2  μm     CWST                                         ______________________________________                                        2.0-0.6   .002         12        50                                           2.0-1.0   .002         9         50                                           2.5-3.5   .003         4.5       66                                           5.6-7.1   .006         3.0       66                                           5.2-10.3  .006         2.6       66                                           ______________________________________                                    

Each of the examples used a single unit of blood donated by a volunteer.The unit of blood was collected in a blood collection set configured asshown in FIG. 1 (without the optional red cell barrier medium assembly),the collection bag of the set having been pre-filled with 63 ml of CPDAanticoagulant. The volume of blood collected from the various donors isshown in Column 1 of Table II. The collection set was positioned intothe centrifuge bucket of FIG. 4 in accordance with customary blood bankpractice, except that the PRC filter was assembled to the bracket, whichin turn was mounted on the upper portion of the centrifuge bucket, thussecuring the PRC filter outside and above the centrifuge bucket.

The centrifuge was then rotated at a speed that developed 2280 G (at thebottom of the bucket) for 3 minutes, sufficient to cause the red bloodcells to sediment into the lower portion of the collection bag. Thebracket was then removed and the collection bag was transferred, withcare to avoid disturbing its contents, to a plasma extractor, which wasspring biased to develop a pressure of about 90 mm Hg. Breaking the sealconnecting the collection bag to the PRP filter and then breaking theseal connecting the PRP filter to the satellite bag permitted the PRPsupernatant layer to flow from the collection bag through the PRP filterinto the satellite bag. As the PRP exited, the interface between the PRCand PRP rose in the collection bag, with flow continuing for about 10 to20 minutes until all of the PRP had passed through the PRP filter, atwhich time the flow terminated abruptly and automatically as the leadingedge of the PRC layer reached the PRP filter. The tubing was thenclamped adjacent to the collection bag, and adjacent to the satellitebag, and the tubing between the two clamps and the PRP filter was cut.The PRP collected in the satellite bag was then processed using normalblood bank procedures to produce leucocyte-depleted PC and plasma. Thevolumes of PC and plasma are shown in Table II along with the number ofresidual leucocytes in the PC.

The collection bag, now containing only the sedimented red cells, wasremoved from the plasma extractor, and 100 ml of AS3 nutrient solution,which had been preplaced in the other satellite bag, was transferredinto the collection bag through the PRC filter. The contents of thecollection bag were then thoroughly mixed. With about 120 mm Hg pressureapplied by gravity head, the PRC in the collection bag was nextleucocyte-depleted by passing it through the PRC filter to the satellitebag. The PRC was now available for transfusion into a patient asrequired. The volume, hematocrits, and the residual leucocyte counts inthe PRC are listed in Table II.

The leucocyte counts presented in the table reflect the sensitivity ofthe methods used for assaying the number of leucocytes residual in thePRC and in the PC effluents. No leucocytes were in fact detected in theleucocyte depleted effluents of any of the examples. Parallelexperiments using more sensitive (but more laborious) assay methodsindicate that the leucocyte depletion efficiencies were about ten to onehundred times better than is indicated by the data presented in thetable.

                                      TABLE II                                    __________________________________________________________________________               Test 1                                                                              Test 2                                                                              Test 3                                                                              Test 4                                                                              Test 5                                     __________________________________________________________________________    Whole Blood                                                                              407   387   399   410   410                                        Collected (mL)                                                                Whole Blood Hct                                                                          45    42.5  40    41    38.5                                       (%)                                                                           PRP Filtration                                                                           16    11    14    15    19                                         Time (min.)                                                                   PRP Volume (mL)                                                                          211   173   196   177   232                                        PC Volume (mL)                                                                           47    52    49    69    61                                         Residual WBC-PC*                                                                         <1.0 × 10.sup.5                                                               <1.1 × 10.sup.5                                                               <1.1 × 10.sup.5                                                               <1.5 × 10.sup.5                                                               <1.3 × 10.sup.5                      PRC Filtration                                                                           15    18    11    11    12                                         Time (min.)                                                                   PRC Volume (mL)                                                                          285   318   301   306   288                                        PRC Hct (%)                                                                              64.5  67    51    52.5  60.5                                       Residual WBC-PRC*                                                                        <7.3 × 10.sup.6                                                               <8.0 × 10.sup.6                                                               <7.5 × 10.sup.6                                                               <7.7 × 10.sup.6                                                               <7.2 × 10.sup.                       __________________________________________________________________________                                       6                                           *per unit                                                                

While the invention has been described in some detail by way ofillustration and example, it should be understood that the invention issusceptible to various modifications and alternative forms, and is notrestricted to the specific embodiments set forth in the Examples. Itshould be understood that these specific embodiments are not intended tolimit the invention but, on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

What is claimed is:
 1. A method for processing a biological fluidcomprising:expressing a biological fluid from a first container to afirst porous medium comprising a red cell barrier medium; and expressinga biological fluid from the first container to a second porous medium.2. The method of claim 1 further comprising expressing the biologicalfluid in the first container to the first porous medium until the redcell barrier medium is blocked, and expressing the biological fluid inthe first container to the second porous medium after the red cellbarrier medium is blocked.
 3. The method of claim 1 further comprisingexpressing the biological fluid from the first container to the firstporous medium and expressing the biological fluid from the firstcontainer to a second porous medium concurrently.
 4. The method of claim1 further comprising expressing the biological fluid from the firstcontainer to the first porous medium and expressing the biological fluidfrom the first container to a second porous medium sequentially.
 5. Themethod of claim 1 further comprising passing the biological fluidthrough a red cell barrier medium and then through a leucocyte depletionmedium.
 6. The method of claim 1 further comprising introducing gasthrough a gas inlet and expelling gas through a gas outlet.
 7. Themethod of claim 1 further comprising processing the biological fluidwithin about 8 hours of collection.
 8. The method of claim 1 furthercomprising processing the biological fluid in a closed sterile system.9. The method of claim 1 wherein expressing the biological fluid fromthe first container to the first porous medium comprises expressing asupernatant layer of the biological fluid; andexpressing the biologicalfluid from the first container to the second porous medium comprisesexpressing a sediment layer of the biological fluid.
 10. The method ofclaim 9 further comprising expressing the supernatant layer of thebiological fluid to the first porous medium until the red cell barriermedium is blocked, and expressing the sediment layer of the biologicalfluid to the second porous medium after the red cell barrier medium isblocked.
 11. A method for processing whole blood comprising:centrifugingwhole blood; expressing a supernatant layer of the centrifuged bloodthrough a first porous medium comprising a red cell barrier medium; andexpressing a sediment layer of the centrifuged blood through a secondporous medium comprising a leucocyte depletion medium.
 12. The method ofclaim 11 further comprising expressing the supernatant layer of thecentrifuged blood through the first porous medium until the red cellbarrier medium is blocked, and expressing the sediment layer of thecentrifuged blood through the second porous medium after the red cellbarrier medium is blocked.
 13. The method of claim 11 further comprisingexpressing the supernatant layer of the centrifuged blood through thefirst porous medium and expressing the sediment layer of the centrifugedblood through the second porous medium concurrently.
 14. The method ofclaim 11 further comprising expressing the supernatant layer of thecentrifuged blood through the first porous medium and expressing thesediment layer of the centrifuged blood through the second porous mediumsimultaneously.
 15. The method of claim 11 further comprising passingthe supernatant layer through a leucocyte depletion medium downstream ofthe first porous medium.
 16. The method of claim 15 further comprisingpassing the supernatant layer through a separation medium downstream ofthe leucocyte depletion medium.
 17. A method for processing whole bloodcomprising:centrifuging whole blood; expressing a supernatant layer ofthe centrifuged blood through a first porous medium comprising a redcell barrier medium and a leucocyte depletion medium; and expressing asediment layer of the centrifuged blood through a second porous mediumcomprising a leucocyte depletion medium.
 18. A method for processingwhole blood comprising:centrifuging whole blood; expressing asupernatant layer of the centrifuged blood through a first porous mediumcomprising a red cell barrier medium until the red cell barrier mediumis blocked; and then, passing the supernatant layer through a separationmedium.
 19. The method of claim 21 wherein passing the supernatant layerthrough a separation medium comprises separating the supernatant layerinto one or more components.
 20. The method of claim 22 furthercomprising collecting the one or more components of the supernatantlayer in at least one container.
 21. A biological fluid processingsystem comprising:a first container; a first porous medium comprising ared cell barrier medium communicating with the first container, anddefining a first flow path; and a second porous medium comprising aleucocyte depletion medium communicating with the first container, anddefining a second flow path.
 22. The biological fluid processing systemof claim 21 further comprising a second container communicating with thesecond porous medium.
 23. The biological fluid processing system ofclaim 21 further comprising a leucocyte depletion medium downstream ofthe first porous medium.
 24. The biological fluid processing system ofclaim 21 further comprising at least one gas inlet and at least one gasoutlet.
 25. The biological fluid processing system of claim 14 whereinthe system comprises a closed sterile system.
 26. A biological fluidprocessing system comprising:a first container; a first porous mediumcomprising a red cell barrier medium communicating with the firstcontainer and defining a first flow path; a second container downstreamof, and communicating with, the first container and defining a secondflow path.
 27. A biological fluid processing system comprising:a firstcontainer; a first porous medium comprising a red cell barrier mediumcommunicating with the first container; and a second porous mediumcomprising a leucocyte depletion medium communicating with the firstcontainer independently of the first porous medium.
 28. The system ofclaim 27, wherein the first porous medium and the second porous mediumindependently communicate with the first container by conduits.
 29. Abiological fluid processing system comprising:a first container; a firstporous medium comprising a porous red cell barrier medium having a CWSTof greater than about 70 dynes/cm communicating with the firstcontainer; and a second porous medium comprising a porous leucocytedepletion medium having a CWST of greater than about 53 dynes/cmcommunicating with the first container.
 30. A biological fluidprocessing system comprising:a first container; a first porous mediumcomprising a red cell barrier medium and a leucocyte depletion mediumcommunicating with the first container, and defining a first flow path;and a second porous medium comprising a leucocyte depletion mediumcommunicating with the first container, and defining a second flow path.31. A biological fluid processing system comprising:a first container; afirst porous medium comprising a red cell barrier medium communicatingwith the first container; and a separation medium downstream of, andcommunicating with, the first porous medium.
 32. A biological fluidprocessing system comprising:a first container; a first porous mediumcomprising a red cell barrier medium communicating with the firstcontainer; a second porous medium comprising a leucocyte depletionmedium communicating with the first container independently of the firstporous medium; a third container communicating with the second porousmedium; a second container communicating with the first porous medium;and, a fourth container communicating with the second container.